Motility and Segregation of Hsp104-Associated Protein Aggregates in Budding Yeast

PubMed Central

Zhou, Chuankai; Slaughter, Brian D.; Unruh, Jay R.; Eldakak, Amr; Rubinstein, Boris; Li, Rong

2011-01-01

SUMMARY During yeast cell division, aggregates of damaged proteins are segregated asymmetrically between the bud and the mother. It is thought that protein aggregates are cleared from the bud via actin cable-based retrograde transport toward the mother, and that Bni1p formin regulates this transport. Here we examined the dynamics of Hsp104-associated protein aggregates by video microscopy, particle tracking and image correlation analysis. We show that protein aggregates undergo random walk without directional bias. Clearance of heat-induced aggregates from the bud does not depend on formin proteins but occurs mostly through dissolution via Hsp104p chaperon. Aggregates formed naturally in aged cells also exhibit random walk but do not dissolve during observation. Although our data does not disagree with a role for actin or cell polarity in aggregate segregation, modeling suggests that their asymmetric inheritance can be a predictable outcome of aggregates' slow diffusion and the geometry of yeast cells. PMID:22118470

High-Throughput Multiplexed Quantitation of Protein Aggregation and Cytotoxicity in a Huntington’s Disease Model

PubMed Central

Titus, Steven A; Southall, Noel; Marugan, Juan; Austin, Christopher P; Zheng, Wei

2012-01-01

A hallmark of Huntington’s disease is the presence of a large polyglutamine expansion in the first exon of the Huntingtin protein and the propensity of protein aggregation by the mutant proteins. Aberrant protein aggregation also occurs in other polyglutamine expansion disorders, as well as in other neurodegenerative diseases including Parkinson’s, Alzheimer’s, and prion diseases. However, the pathophysiological role of these aggregates in the cell death that characterizes the diseases remains unclear. Identification of small molecule probes that modulate protein aggregation and cytotoxicity caused by aggregated proteins may greatly facilitate the studies on pathogenesis of these diseases and potentially lead to development of new therapies. Based on a detergent insoluble property of the Huntingtin protein aggregates, we have developed a homogenous assay to rapidly quantitate the levels of protein aggregates in a cellular model of Huntington’s disease. The protein aggregation assay has also been multiplexed with a protease release assay for the measurement of cytotoxicity resulting from aggregated proteins in the same cells. Through a testing screen of a compound library, we have demonstrated that this multiplexed cytotoxicity and protein aggregation assay has ability to identify active compounds that prevent cell death and/or modulate protein aggregation in cells of the Huntington’s disease model. Therefore, this multiplexed screening approach is also useful for development of high-throughput screening assays for other neurodegenerative diseases involving protein aggregation. PMID:23346268

A549 lung epithelial cells grown as three-dimensional aggregates: alternative tissue culture model for Pseudomonas aeruginosa pathogenesis.

PubMed

Carterson, A J; Höner zu Bentrup, K; Ott, C M; Clarke, M S; Pierson, D L; Vanderburg, C R; Buchanan, K L; Nickerson, C A; Schurr, M J

2005-02-01

A three-dimensional (3-D) lung aggregate model was developed from A549 human lung epithelial cells by using a rotating-wall vessel bioreactor to study the interactions between Pseudomonas aeruginosa and lung epithelial cells. The suitability of the 3-D aggregates as an infection model was examined by immunohistochemistry, adherence and invasion assays, scanning electron microscopy, and cytokine and mucoglycoprotein production. Immunohistochemical characterization of the 3-D A549 aggregates showed increased expression of epithelial cell-specific markers and decreased expression of cancer-specific markers compared to their monolayer counterparts. Immunohistochemistry of junctional markers on A549 3-D cells revealed that these cells formed tight junctions and polarity, in contrast to the cells grown as monolayers. Additionally, the 3-D aggregates stained positively for the production of mucoglycoprotein while the monolayers showed no indication of staining. Moreover, mucin-specific antibodies to MUC1 and MUC5A bound with greater affinity to 3-D aggregates than to the monolayers. P. aeruginosa attached to and penetrated A549 monolayers significantly more than the same cells grown as 3-D aggregates. Scanning electron microscopy of A549 cells grown as monolayers and 3-D aggregates infected with P. aeruginosa showed that monolayers detached from the surface of the culture plate postinfection, in contrast to the 3-D aggregates, which remained attached to the microcarrier beads. In response to infection, proinflammatory cytokine levels were elevated for the 3-D A549 aggregates compared to monolayer controls. These findings suggest that A549 lung cells grown as 3-D aggregates may represent a more physiologically relevant model to examine the interactions between P. aeruginosa and the lung epithelium during infection.

Automatic analysis of microscopic images of red blood cell aggregates

NASA Astrophysics Data System (ADS)

Menichini, Pablo A.; Larese, Mónica G.; Riquelme, Bibiana D.

2015-06-01

Red blood cell aggregation is one of the most important factors in blood viscosity at stasis or at very low rates of flow. The basic structure of aggregates is a linear array of cell commonly termed as rouleaux. Enhanced or abnormal aggregation is seen in clinical conditions, such as diabetes and hypertension, producing alterations in the microcirculation, some of which can be analyzed through the characterization of aggregated cells. Frequently, image processing and analysis for the characterization of RBC aggregation were done manually or semi-automatically using interactive tools. We propose a system that processes images of RBC aggregation and automatically obtains the characterization and quantification of the different types of RBC aggregates. Present technique could be interesting to perform the adaptation as a routine used in hemorheological and Clinical Biochemistry Laboratories because this automatic method is rapid, efficient and economical, and at the same time independent of the user performing the analysis (repeatability of the analysis).

Cryopreservation of pluripotent stem cell aggregates in defined protein-free formulation.

PubMed

Sart, Sébastien; Ma, Teng; Li, Yan

2013-01-01

Cultivation of undifferentiated pluripotent stem cells (PSCs) as aggregates has emerged as an efficient culture configuration, enabling rapid and controlled large scale expansion. Aggregate-based PSC cryopreservation facilitates the integrated process of cell expansion and cryopreservation, but its feasibility has not been demonstrated. The goals of current study are to assess the suitability of cryopreserving intact mouse embryonic stem cell (mESC) aggregates and investigate the effects of aggregate size and the formulation of cryopreservation solution on mESC survival and recovery. The results demonstrated the size-dependent cell survival and recovery of intact aggregates. In particular, the generation of reactive oxygen species (ROS) and caspase activation were reduced for small aggregates (109 ± 55 μm) compared to medium (245 ± 77 μm) and large (365 ± 141 μm) ones, leading to the improved cell recovery. In addition, a defined protein-free formulation was tested and found to promote the aggregate survival, eliminating the cell exposure to animal serum. The cryopreserved aggregates also maintained the pluripotent markers and the differentiation capacity into three-germ layers after thawing. In summary, the cryopreservation of small PSC aggregates in a defined protein-free formulation was shown to be a suitable approach toward a fully integrated expansion and cryopreservation process at large scale. Copyright © 2012 American Institute of Chemical Engineers (AIChE).

Characterization of circulating tumor cell aggregates identified in patients with epithelial tumors

NASA Astrophysics Data System (ADS)

Cho, Edward H.; Wendel, Marco; Luttgen, Madelyn; Yoshioka, Craig; Marrinucci, Dena; Lazar, Daniel; Schram, Ethan; Nieva, Jorge; Bazhenova, Lyudmila; Morgan, Alison; Ko, Andrew H.; Korn, W. Michael; Kolatkar, Anand; Bethel, Kelly; Kuhn, Peter

2012-02-01

Circulating tumor cells (CTCs) have been implicated as a population of cells that may seed metastasis and venous thromboembolism (VTE), two major causes of mortality in cancer patients. Thus far, existing CTC detection technologies have been unable to reproducibly detect CTC aggregates in order to address what contribution CTC aggregates may make to metastasis or VTE. We report here an enrichment-free immunofluorescence detection method that can reproducibly detect and enumerate homotypic CTC aggregates in patient samples. We identified CTC aggregates in 43% of 86 patient samples. The fraction of CTC aggregation was investigated in blood draws from 24 breast, 14 non-small cell lung, 18 pancreatic, 15 prostate stage IV cancer patients and 15 normal blood donors. Both single CTCs and CTC aggregates were measured to determine whether differences exist in the physical characteristics of these two populations. Cells contained in CTC aggregates had less area and length, on average, than single CTCs. Nuclear to cytoplasmic ratios between single CTCs and CTC aggregates were similar. This detection method may assist future studies in determining which population of cells is more physically likely to contribute to metastasis and VTE.

Compartmentalization of superoxide dismutase 1 (SOD1G93A) aggregates determines their toxicity

PubMed Central

Weisberg, Sarah J.; Lyakhovetsky, Roman; Werdiger, Ayelet-chen; Gitler, Aaron D.; Soen, Yoav; Kaganovich, Daniel

2012-01-01

Neurodegenerative diseases constitute a class of illnesses marked by pathological protein aggregation in the brains of affected individuals. Although these disorders are invariably characterized by the degeneration of highly specific subpopulations of neurons, protein aggregation occurs in all cells, which indicates that toxicity arises only in particular cell biological contexts. Aggregation-associated disorders are unified by a common cell biological feature: the deposition of the culprit proteins in inclusion bodies. The precise function of these inclusions remains unclear. The starting point for uncovering the origins of disease pathology must therefore be a thorough understanding of the general cell biological function of inclusions and their potential role in modulating the consequences of aggregation. Here, we show that in human cells certain aggregate inclusions are active compartments. We find that toxic aggregates localize to one of these compartments, the juxtanuclear quality control compartment (JUNQ), and interfere with its quality control function. The accumulation of SOD1G93A aggregates sequesters Hsp70, preventing the delivery of misfolded proteins to the proteasome. Preventing the accumulation of SOD1G93A in the JUNQ by enhancing its sequestration in an insoluble inclusion reduces the harmful effects of aggregation on cell viability. PMID:22967507

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

Levin, Johannes; German Center for Neurodegenerative Diseases – DZNE, Site Munich, Feodor-Lynen-Str. 17, 81377 Munich; Hillmer, Andreas S.

Synucleinopathies such as dementia with Lewy bodies or Parkinson’s disease are characterized by intracellular deposition of pathologically aggregated α-synuclein. The details of the molecular pathogenesis of PD and especially the conditions that lead to intracellular aggregation of α-synuclein and the role of these aggregates in cell death remain unknown. In cell free in vitro systems considerable knowledge about the aggregation processes has been gathered. In comparison, the knowledge about these aggregation processes in cells is far behind. In cells α-synuclein aggregates can be toxic. However, the crucial particle species responsible for decisive steps in pathogenesis such as seeding a continuing aggregationmore » process and triggering cell death remain to be identified. In order to understand the complex nature of intracellular α-synuclein aggregate formation, we analyzed fluorescent particles formed by venus and α-synuclein-venus fusion proteins and α-synuclein-hemi-venus fusion proteins derived from gently lyzed cells. With these techniques we were able to identify and characterize α-synuclein oligomers formed in cells. Especially the use of α-synuclein-hemi-venus fusion proteins enabled us to identify very small α-synuclein oligomers with high sensitivity. Furthermore, we were able to study the molecular effect of heat shock protein 70, which is known to inhibit α-synuclein aggregation in cells. Heat shock protein 70 does not only influence the size of α-synuclein oligomers, but also their quantity. In summary, this approach based on fluorescence single particle spectroscopy, that is suited for high throughput measurements, can be used to detect and characterize intracellularly formed α-synuclein aggregates and characterize the effect of molecules that interfere with α-synuclein aggregate formation. - Highlights: • Single particle spectroscopy detects intracellular formed α-synuclein aggregates. • Fusion proteins allow detection of protein aggregates at the oligomer level. • The technique detects molecules inhibiting α-synuclein aggregate formation. • Single particle spectroscopy is suited for high throughput measurements.« less

Coupling of aggregation and immunogenicity in biotherapeutics: T- and B-cell immune epitopes may contain aggregation-prone regions.

PubMed

Kumar, Sandeep; Singh, Satish K; Wang, Xiaoling; Rup, Bonita; Gill, Davinder

2011-05-01

Biotherapeutics, including recombinant or plasma-derived human proteins and antibody-based molecules, have emerged as an important class of pharmaceuticals. Aggregation and immunogenicity are among the major bottlenecks during discovery and development of biotherapeutics. Computational tools that can predict aggregation prone regions as well as T- and B-cell immune epitopes from protein sequence and structure have become available recently. Here, we describe a potential coupling between aggregation and immunogenicity: T-cell and B-cell immune epitopes in therapeutic proteins may contain aggregation-prone regions. The details of biological mechanisms behind this observation remain to be understood. However, our observation opens up an exciting potential for rational design of de-immunized novel, as well as follow on biotherapeutics with reduced aggregation propensity.

Inhibition of Human Amylin Aggregation and Cellular Toxicity by Lipoic Acid and Ascorbic Acid.

PubMed

Azzam, Sarah Kassem; Jang, Hyunwoo; Choi, Myung Chul; Alsafar, Habiba; Lukman, Suryani; Lee, Sungmun

2018-04-30

More than 30 human degenerative diseases result from protein aggregation such as Alzheimer's disease (AD) and type 2 diabetes mellitus (T2DM). Islet amyloid deposits, a hallmark in T2DM, are found in pancreatic islets of more than 90 % of T2DM patients. An association between amylin aggregation and reduction in β-cell mass was also established by post-mortem studies. A strategy in preventing protein aggregation-related disorders is to inhibit the protein aggregation and associated toxicity. In this study we demonstrated that two inhibitors, lipoic acid and ascorbic acid, significantly inhibited amylin aggregation. Compared to amylin (15 μM) as 100 %, lipoic acid and ascorbic acid reduced amylin fibril formation to 42.1 ± 17.2 % and 42.9 ± 12.8 % respectively, which is confirmed by fluorescence and TEM images. In cell viability tests, both inhibitors protected RIN-m5f β-cells from the toxicity of amylin aggregates. At 10:1 molar ratio of lipoic acid to amylin, lipoic acid with amylin increased the cell viability to 70.3 %, whereas only 42.8 % RIN-m5f β-cells survived in amylin aggregates. For ascorbic acid, an equimolar ratio achieved the highest cell viability of 63.3 % as compared to 42.8 % with amylin aggregates only. Docking results showed that lipoic acid and ascorbic acid physically interact with amylin amyloidogenic region (residues Ser20-Ser29) via hydrophobic interactions; hence reducing aggregation levels. Therefore, lipoic acid and ascorbic acid prevented amylin aggregation via hydrophobic interactions, which resulted in the prevention of cell toxicity in vitro.

Dielectric spectroscopy platform to measure MCF10A epithelial cell aggregation as a model for spheroidal cell cluster analysis.

PubMed

Heileman, K L; Tabrizian, M

2017-05-02

3-Dimensional cell cultures are more representative of the native environment than traditional cell cultures on flat substrates. As a result, 3-dimensional cell cultures have emerged as a very valuable model environment to study tumorigenesis, organogenesis and tissue regeneration. Many of these models encompass the formation of cell aggregates, which mimic the architecture of tumor and organ tissue. Dielectric impedance spectroscopy is a non-invasive, label free and real time technique, overcoming the drawbacks of established techniques to monitor cell aggregates. Here we introduce a platform to monitor cell aggregation in a 3-dimensional extracellular matrix using dielectric spectroscopy. The MCF10A breast epithelial cell line serves as a model for cell aggregation. The platform maintains sterile conditions during the multi-day assay while allowing continuous dielectric spectroscopy measurements. The platform geometry optimizes dielectric measurements by concentrating cells within the electrode sensing region. The cells show a characteristic dielectric response to aggregation which corroborates with finite element analysis computer simulations. By fitting the experimental dielectric spectra to the Cole-Cole equation, we demonstrated that the dispersion intensity Δε and the characteristic frequency f c are related to cell aggregate growth. In addition, microscopy can be performed directly on the platform providing information about cell position, density and morphology. This platform could yield many applications for studying the electrophysiological activity of cell aggregates.

Lysosomal enzyme cathepsin B enhances the aggregate forming activity of exogenous α-synuclein fibrils.

PubMed

Tsujimura, Atsushi; Taguchi, Katsutoshi; Watanabe, Yoshihisa; Tatebe, Harutsugu; Tokuda, Takahiko; Mizuno, Toshiki; Tanaka, Masaki

2015-01-01

The formation of intracellular aggregates containing α-synuclein (α-Syn) is one of the key steps in the progression of Parkinson's disease and dementia with Lewy bodies. Recently, it was reported that pathological α-Syn fibrils can undergo cell-to-cell transmission and form Lewy body-like aggregates. However, little is known about how they form α-Syn aggregates from fibril seeds. Here, we developed an assay to study the process of aggregate formation using fluorescent protein-tagged α-Syn-expressing cells and examined the aggregate forming activity of exogenous α-Syn fibrils. α-Syn fibril-induced formation of intracellular aggregates was suppressed by a cathepsin B specific inhibitor, but not by a cathepsin D inhibitor. α-Syn fibrils pretreated with cathepsin B in vitro enhanced seeding activity in cells. Knockdown of cathepsin B also reduced fibril-induced aggregate formation. Moreover, using LAMP-1 immunocytochemistry and live-cell imaging, we observed that these aggregates initially occurred in the lysosome. They then rapidly grew larger and moved outside the boundary of the lysosome within one day. These results suggest that the lysosomal protease cathepsin B is involved in triggering intracellular aggregate formation by α-Syn fibrils. Copyright © 2015. Published by Elsevier Inc.

Aggregation via the Red, Dry, and Rough Morphotype Is Not a Virulence Adaptation in Salmonella enterica Serovar Typhimurium▿

PubMed Central

White, A. P.; Gibson, D. L.; Grassl, G. A.; Kay, W. W.; Finlay, B. B.; Vallance, B. A.; Surette, M. G.

2008-01-01

The Salmonella rdar (red, dry, and rough) morphotype is an aggregative and resistant physiology that has been linked to survival in nutrient-limited environments. Growth of Salmonella enterica serovar Typhimurium was analyzed in a variety of nutrient-limiting conditions to determine whether aggregation would occur at low cell densities and whether the rdar morphotype was involved in this process. The resulting cultures consisted of two populations of cells, aggregated and nonaggregated, with the aggregated cells preferentially displaying rdar morphotype gene expression. The two groups of cells could be separated based on the principle that aggregated cells were producing greater amounts of thin aggregative fimbriae (Tafi or curli). In addition, the aggregated cells retained some physiological characteristics of the rdar morphotype, such as increased resistance to sodium hypochlorite. Competitive infection experiments in mice showed that nonaggregative ΔagfA cells outcompeted rdar-positive wild-type cells in all tissues analyzed, indicating that aggregation via the rdar morphotype was not a virulence adaptation in Salmonella enterica serovar Typhimurium. Furthermore, in vivo imaging experiments showed that Tafi genes were not expressed during infection but were expressed once Salmonella was passed out of the mice into the feces. We hypothesize that the primary role of the rdar morphotype is to enhance Salmonella survival outside the host, thereby aiding in transmission. PMID:18195033

Transplantation of co-aggregates of Sertoli cells and islet cells into liver without immunosuppression.

PubMed

Takemoto, Naohiro; Liu, Xibao; Takii, Kento; Teramura, Yuji; Iwata, Hiroo

2014-02-15

Transplantation of islets of Langerhans (islets) was used to treat insulin-dependent diabetes mellitus. However, islet grafts must be maintained by administration of immunosuppressive drugs, which can lead to complications in the long term. An approach that avoids immunosuppressive drug use is desirable. Co-aggregates of Sertoli cells and islet cells from BALB/c mice that were prepared by the hanging drop method were transplanted into C57BL/6 mouse liver through the portal vein as in human clinical islet transplantation. The core part of the aggregates contained mainly Sertoli cells, and these cells were surrounded by islet cells. The co-aggregates retained the functions of both Sertoli and islet cells. When 800 co-aggregates were transplanted into seven C57BL/6 mice via the portal vein, six of seven recipient mice demonstrated quasi-normoglycemia for more than 100 days. The hanging drop method is suitable for preparing aggregates of Sertoli and islet cells for transplantation. Notably, transplantation of these allogeneic co-aggregates into mice with chemically induced diabetes via the portal vein resulted in long-term graft survival without systemic immunosuppression.

Describing Myxococcus xanthus Aggregation Using Ostwald Ripening Equations for Thin Liquid Films

PubMed Central

Bahar, Fatmagül; Pratt-Szeliga, Philip C.; Angus, Stuart; Guo, Jiaye; Welch, Roy D.

2014-01-01

When starved, a swarm of millions of Myxococcus xanthus cells coordinate their movement from outward swarming to inward coalescence. The cells then execute a synchronous program of multicellular development, arranging themselves into dome shaped aggregates. Over the course of development, about half of the initial aggregates disappear, while others persist and mature into fruiting bodies. This work seeks to develop a quantitative model for aggregation that accurately simulates which will disappear and which will persist. We analyzed time-lapse movies of M. xanthus development, modeled aggregation using the equations that describe Ostwald ripening of droplets in thin liquid films, and predicted the disappearance and persistence of aggregates with an average accuracy of 85%. We then experimentally validated a prediction that is fundamental to this model by tracking individual fluorescent cells as they moved between aggregates and demonstrating that cell movement towards and away from aggregates correlates with aggregate disappearance. Describing development through this model may limit the number and type of molecular genetic signals needed to complete M. xanthus development, and it provides numerous additional testable predictions. PMID:25231319

Fibrillar Structure and Charge Determine the Interaction of Polyglutamine Protein Aggregates with the Cell Surface*

PubMed Central

Trevino, R. Sean; Lauckner, Jane E.; Sourigues, Yannick; Pearce, Margaret M.; Bousset, Luc; Melki, Ronald; Kopito, Ron R.

2012-01-01

The pathogenesis of most neurodegenerative diseases, including transmissible diseases like prion encephalopathy, inherited disorders like Huntington disease, and sporadic diseases like Alzheimer and Parkinson diseases, is intimately linked to the formation of fibrillar protein aggregates. It is becoming increasingly appreciated that prion-like intercellular transmission of protein aggregates can contribute to the stereotypical spread of disease pathology within the brain, but the mechanisms underlying the binding and uptake of protein aggregates by mammalian cells are largely uninvestigated. We have investigated the properties of polyglutamine (polyQ) aggregates that endow them with the ability to bind to mammalian cells in culture and the properties of the cell surface that facilitate such uptake. Binding and internalization of polyQ aggregates are common features of mammalian cells and depend upon both trypsin-sensitive and trypsin-resistant saturable sites on the cell surface, suggesting the involvement of cell surface proteins in this process. polyQ aggregate binding depends upon the presence of a fibrillar amyloid-like structure and does not depend upon electrostatic interaction of fibrils with the cell surface. Sequences in the huntingtin protein that flank the amyloid-forming polyQ tract also influence the extent to which aggregates are able to bind to cell surfaces. PMID:22753412

Glyceraldehyde-3-phosphate dehydrogenase aggregation inhibitor peptide: A potential therapeutic strategy against oxidative stress-induced cell death.

PubMed

Itakura, Masanori; Nakajima, Hidemitsu; Semi, Yuko; Higashida, Shusaku; Azuma, Yasu-Taka; Takeuchi, Tadayoshi

2015-11-13

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has multiple functions, including mediating oxidative stress-induced neuronal cell death. This process is associated with disulfide-bonded GAPDH aggregation. Some reports suggest a link between GAPDH and the pathogenesis of several oxidative stress-related diseases. However, the pathological significance of GAPDH aggregation in disease pathogenesis remains unclear due to the lack of an effective GAPDH aggregation inhibitor. In this study, we identified a GAPDH aggregation inhibitor (GAI) peptide and evaluated its biological profile. The decapeptide GAI specifically inhibited GAPDH aggregation in a concentration-dependent manner. Additionally, the GAI peptide did not affect GAPDH glycolytic activity or cell viability. The GAI peptide also exerted a protective effect against oxidative stress-induced cell death in SH-SY5Y cells. This peptide could potentially serve as a tool to investigate GAPDH aggregation-related neurodegenerative and neuropsychiatric disorders and as a possible therapy for diseases associated with oxidative stress-induced cell death. Copyright © 2015 Elsevier Inc. All rights reserved.

Polyglutamine aggregation in Huntington and related diseases.

PubMed

Polling, Saskia; Hill, Andrew F; Hatters, Danny M

2012-01-01

Polyglutamine (polyQ)-expansions in different proteins cause nine neurodegenerative diseases. While polyQ aggregation is a key pathological hallmark of these diseases, how aggregation relates to pathogenesis remains contentious. In this chapter, we review what is known about the aggregation process and how cells respond and interact with the polyQ-expanded proteins. We cover detailed biophysical and structural studies to uncover the intrinsic features of polyQ aggregates and concomitant effects in the cellular environment. We also examine the functional consequences ofpolyQ aggregation and how cells may attempt to intervene and guide the aggregation process.

A critical-like collective state leads to long-range cell communication in Dictyostelium discoideum aggregation

PubMed Central

De Palo, Giovanna; Yi, Darvin; Endres, Robert G.

2017-01-01

The transition from single-cell to multicellular behavior is important in early development but rarely studied. The starvation-induced aggregation of the social amoeba Dictyostelium discoideum into a multicellular slug is known to result from single-cell chemotaxis towards emitted pulses of cyclic adenosine monophosphate (cAMP). However, how exactly do transient, short-range chemical gradients lead to coherent collective movement at a macroscopic scale? Here, we developed a multiscale model verified by quantitative microscopy to describe behaviors ranging widely from chemotaxis and excitability of individual cells to aggregation of thousands of cells. To better understand the mechanism of long-range cell—cell communication and hence aggregation, we analyzed cell—cell correlations, showing evidence of self-organization at the onset of aggregation (as opposed to following a leader cell). Surprisingly, cell collectives, despite their finite size, show features of criticality known from phase transitions in physical systems. By comparing wild-type and mutant cells with impaired aggregation, we found the longest cell—cell communication distance in wild-type cells, suggesting that criticality provides an adaptive advantage and optimally sized aggregates for the dispersal of spores. PMID:28422986

A cell-based assay for aggregation inhibitors as therapeutics of polyglutamine-repeat disease and validation in Drosophila

NASA Astrophysics Data System (ADS)

Apostol, Barbara L.; Kazantsev, Alexsey; Raffioni, Simona; Illes, Katalin; Pallos, Judit; Bodai, Laszlo; Slepko, Natalia; Bear, James E.; Gertler, Frank B.; Hersch, Steven; Housman, David E.; Marsh, J. Lawrence; Michels Thompson, Leslie

2003-05-01

The formation of polyglutamine-containing aggregates and inclusions are hallmarks of pathogenesis in Huntington's disease that can be recapitulated in model systems. Although the contribution of inclusions to pathogenesis is unclear, cell-based assays can be used to screen for chemical compounds that affect aggregation and may provide therapeutic benefit. We have developed inducible PC12 cell-culture models to screen for loss of visible aggregates. To test the validity of this approach, compounds that inhibit aggregation in the PC12 cell-based screen were tested in a Drosophila model of polyglutamine-repeat disease. The disruption of aggregation in PC12 cells strongly correlates with suppression of neuronal degeneration in Drosophila. Thus, the engineered PC12 cells coupled with the Drosophila model provide a rapid and effective method to screen and validate compounds.

Fluorescence dye-based detection of mAb aggregates in CHO culture supernatants.

PubMed

Paul, Albert Jesuran; Schwab, Karen; Prokoph, Nina; Haas, Elena; Handrick, René; Hesse, Friedemann

2015-06-01

Product yields, efficacy, and safety of monoclonal antibodies (mAbs) are reduced by the formation of higher molecular weight aggregates during upstream processing. In-process characterization of mAb aggregate formation is a challenge since there is a lack of a fast detection method to identify mAb aggregates in cell culture. In this work, we present a rapid method to characterize mAb aggregate-containing Chinese hamster ovary (CHO) cell culture supernatants. The fluorescence dyes thioflavin T (ThT) and 4-4-bis-1-phenylamino-8-naphthalene sulfonate (Bis-ANS) enabled the detection of soluble as well as large mAb aggregates. Partial least square (PLS) regression models were used to evaluate the linearity of the dye-based mAb aggregate detection in buffer down to a mAb aggregate concentration of 2.4 μg mL(-1). Furthermore, mAb aggregates were detected in bioprocess medium using Bis-ANS and ThT. Dye binding to aggregates was stable for 60 min, making the method robust and reliable. Finally, the developed method using 10 μmol L(-1) Bis-ANS enabled discrimination between CHO cell culture supernatants containing different levels of mAb aggregates. The method can be adapted for high-throughput screening, e.g., to screen for cell culture conditions influencing mAb product quality, and hence can contribute to the improvement of production processes of biopharmaceuticals in mammalian cell culture.

Evidence for Compression of Escherichia coli K12 Cells under the Effect of TiO₂ Nanoparticles.

PubMed

Zhukova, Lyudmila V

2015-12-16

It has been shown that treatment with titanium dioxide nanoparticles (TiO2 NPs) combined with near-ultraviolet (UV-A) irradiation or in certain dark conditions reduced the numbers of various microorganisms, but the mechanism of this effect remains unclear. In this study to further clarify the mechanism of the antibacterial effect of TiO2 NPs the physiological state of E. coli K12 cells was estimated after incubation with the NPs (0.2 g/L) for different periods of time, with or without UV-A irradiation. Cell incubation with TiO2 NPs, combined or not combined with UV-A irradiation, showed that inactive cells were located only within cell aggregates formed after incubation with TiO2 NPs and that the larger the aggregate, the greater the number of such cells. When the formation of large aggregates was prevented, exposure to NPs under UV-A irradiation failed to result in cell inactivation. A comparative analysis of fluorescence and optical microscopic images of the same aggregates showed that the location of inactivated cells coincided with the zone of increased optical density within the aggregate. After treatment with TiO2 NPs under UV-A for 30, 60, or 120 min cells within the aggregates were the first to be inactivated. Cells on which NPs irradiated more strongly (at the periphery of large aggregates and single) remained active for a longer time than cells within the aggregates. As the time of treatment increased, so did the degree of cell compaction, with some zones of the aggregates eventually transforming into an acellular mass. After UV-A irradiation the cell aggregates spontaneously moved toward each other and gradually fused into larger structures, indicating that such exposure enhanced mutual attraction of cells treated with the NPs. Present study provides evidence for hypothesis that bacterial cells covered with TiO2 NPs are inactivated due to their mutual attraction and consequent compression.

Long-term Culture of Human iPS Cell-derived Telencephalic Neuron Aggregates on Collagen Gel.

PubMed

Oyama, Hiroshi; Takahashi, Koji; Tanaka, Yoshikazu; Takemoto, Hiroshi; Haga, Hisashi

2018-01-01

It takes several months to form the 3-dimensional morphology of the human embryonic brain. Therefore, establishing a long-term culture method for neuronal tissues derived from human induced pluripotent stem (iPS) cells is very important for studying human brain development. However, it is difficult to keep primary neurons alive for more than 3 weeks in culture. Moreover, long-term adherent culture to maintain the morphology of telencephalic neuron aggregates induced from human iPS cells is also difficult. Although collagen gel has been widely used to support long-term culture of cells, it is not clear whether human iPS cell-derived neuron aggregates can be cultured for long periods on this substrate. In the present study, we differentiated human iPS cells to telencephalic neuron aggregates and examined long-term culture of these aggregates on collagen gel. The results indicated that these aggregates could be cultured for over 3 months by adhering tightly onto collagen gel. Furthermore, telencephalic neuronal precursors within these aggregates matured over time and formed layered structures. Thus, long-term culture of telencephalic neuron aggregates derived from human iPS cells on collagen gel would be useful for studying human cerebral cortex development.Key words: Induced pluripotent stem cell, forebrain neuron, collagen gel, long-term culture.

Light scattering method to measure red blood cell aggregation during incubation

NASA Astrophysics Data System (ADS)

Grzegorzewski, B.; Szołna-Chodór, A.; Baryła, J.; DreŻek, D.

2018-01-01

Red blood cell (RBC) aggregation can be observed both in vivo as well as in vitro. This process is a cause of alterations of blood flow in microvascular network. Enhanced RBC aggregation makes oxygen and nutrients delivery difficult. Measurements of RBC aggregation usually give a description of the process for a sample where the state of a solution and cells is well-defined and the system reached an equilibrium. Incubation of RBCs in various solutions is frequently used to study the effects of the solutions on the RBC aggregation. The aggregation parameters are compared before and after incubation while the detailed changes of the parameters during incubation remain unknown. In this paper we have proposed a method to measure red blood cell aggregation during incubation based on the well-known technique where backscattered light is used to assess the parameters of the RBC aggregation. Couette system consisting of two cylinders is adopted in the method. The incubation is observed in the Couette system. In the proposed method following sequence of rotations is adapted. Two minutes rotation is followed by two minutes stop. In this way we have obtained a time series of back scattered intensity consisting of signals respective for disaggregation and aggregation. It is shown that the temporal changes of the intensity manifest changes of RBC aggregation during incubation. To show the ability of the method to assess the effect of incubation time on RBC aggregation the results are shown for solutions that cause an increase of RBC aggregation as well as for the case where the aggregation is decreased.

Properties and usefulness of aggregates of synovial mesenchymal stem cells as a source for cartilage regeneration

PubMed Central

2012-01-01

Introduction Transplantation of mesenchymal stem cells (MSCs) derived from synovium is a promising therapy for cartilage regeneration. For clinical application, improvement of handling operation, enhancement of chondrogenic potential, and increase of MSCs adhesion efficiency are needed to achieve a more successful cartilage regeneration with a limited number of MSCs without scaffold. The use of aggregated MSCs may be one of the solutions. Here, we investigated the handling, properties and effectiveness of aggregated MSCs for cartilage regeneration. Methods Human and rabbit synovial MSCs were aggregated using the hanging drop technique. The gene expression changes after aggregation of synovial MSCs were analyzed by microarray and real time RT-PCR analyses. In vitro and in vivo chondrogenic potential of aggregates of synovial MSCs was examined. Results Aggregates of MSCs cultured for three days became visible, approximately 1 mm in diameter and solid and durable by manipulation; most of the cells were viable. Microarray analysis revealed up-regulation of chondrogenesis-related, anti-inflammatory and anti-apoptotic genes in aggregates of MSCs. In vitro studies showed higher amounts of cartilage matrix synthesis in pellets derived from aggregates of MSCs compared to pellets derived from MSCs cultured in a monolayer. In in vivo studies in rabbits, aggregates of MSCs could adhere promptly on the osteochondral defects by surface tension, and stay without any loss. Transplantation of aggregates of MSCs at relatively low density achieved successful cartilage regeneration. Contrary to our expectation, transplantation of aggregates of MSCs at high density failed to regenerate cartilage due to cell death and nutrient deprivation of aggregates of MSCs. Conclusions Aggregated synovial MSCs were a useful source for cartilage regeneration considering such factors as easy preparation, higher chondrogenic potential and efficient attachment. PMID:22676383

Localization of Protein Aggregation in Escherichia coli Is Governed by Diffusion and Nucleoid Macromolecular Crowding Effect

PubMed Central

Coquel, Anne-Sophie; Jacob, Jean-Pascal; Primet, Mael; Demarez, Alice; Dimiccoli, Mariella; Julou, Thomas; Moisan, Lionel

2013-01-01

Aggregates of misfolded proteins are a hallmark of many age-related diseases. Recently, they have been linked to aging of Escherichia coli (E. coli) where protein aggregates accumulate at the old pole region of the aging bacterium. Because of the potential of E. coli as a model organism, elucidating aging and protein aggregation in this bacterium may pave the way to significant advances in our global understanding of aging. A first obstacle along this path is to decipher the mechanisms by which protein aggregates are targeted to specific intercellular locations. Here, using an integrated approach based on individual-based modeling, time-lapse fluorescence microscopy and automated image analysis, we show that the movement of aging-related protein aggregates in E. coli is purely diffusive (Brownian). Using single-particle tracking of protein aggregates in live E. coli cells, we estimated the average size and diffusion constant of the aggregates. Our results provide evidence that the aggregates passively diffuse within the cell, with diffusion constants that depend on their size in agreement with the Stokes-Einstein law. However, the aggregate displacements along the cell long axis are confined to a region that roughly corresponds to the nucleoid-free space in the cell pole, thus confirming the importance of increased macromolecular crowding in the nucleoids. We thus used 3D individual-based modeling to show that these three ingredients (diffusion, aggregation and diffusion hindrance in the nucleoids) are sufficient and necessary to reproduce the available experimental data on aggregate localization in the cells. Taken together, our results strongly support the hypothesis that the localization of aging-related protein aggregates in the poles of E. coli results from the coupling of passive diffusion-aggregation with spatially non-homogeneous macromolecular crowding. They further support the importance of “soft” intracellular structuring (based on macromolecular crowding) in diffusion-based protein localization in E. coli. PMID:23633942

Hemoglobin Aggregation in Single Red Blood Cells of Sickle Cell Anemia

NASA Astrophysics Data System (ADS)

Nishio, Izumi; Tanaka, Toyoichi; Sun, Shao-Tang; Imanishi, Yuri; Tsuyoshi Ohnishi, S.

1983-06-01

A laser light scattering technique was used to observe the extent of hemoglobin aggregation in solitary red blood cells of sickle cell anemia. Hemoglobin aggregation was confirmed in deoxygenated cells. The light scattering technique can also be applied to cytoplasmic studies of any biological cell.

Aggregation Dynamics Using Phase Wave Signals and Branching Patterns

NASA Astrophysics Data System (ADS)

Sakaguchi, Hidetsugu; Kusagaki, Takuma

2016-09-01

The aggregation dynamics of slime mold is studied using coupled equations of phase ϕ and cell concentration n. Phase waves work as tactic signals for aggregation. Branching structures appear during the aggregation. A stationary branching pattern appears like a river network, if cells are uniformly supplied into the system.

Receptors for aggregated IgG on mouse lymphocytes: their presence on thymocytes, thymus-derived, and bone marrow-derived lymphocytes.

PubMed

Anderson, C L; Grey, H M

1974-05-01

An autoradiographic binding assay employing (125)I-labeled heat-aggregated mouse IgG2b myeloma protein (MOPC 141) was used to demonstrate receptors for IgG on 20-45% of Balb/c thymocytes and on 70-80% of splenocytes. Binding could also be shown with heat or BDB aggregates of another IgG2b (MOPC 195), with IgG1 and with human gamma-globulin, but not with aggregated chicken gamma-globulin, IgA, BSA, nor with aggregated Fab fragments of IgG2b. Optimum binding was obtained at 37 degrees C. Detection of binding was dependent upon aggregate size with complexes of more than 100 IgG molecules being optimal, aggregates of 6-25 detecting splenocytes but not thymocytes, and aggregates of less than 6 binding to a negligible extent. Comparison of grain counts on various cell types showed mastocytoma cells (P815) and macrophages averaging 40-50 grains/cell/day, allogeneically activated thymocytes 20-30, splenocytes 2-3, L5178 lymphoma cells 1, and positive thymocytes 0.6 grains/cell/day. Double labeling experiments for surface Ig, theta-antigen, and agg IgG receptor on mouse spleen cells indicated that a relatively high density of receptor was present on about 80% of B cells, 30% of T cells, and 60% of SIg(-), theta(-), null cells.

Data-driven modeling reveals cell behaviors controlling self-organization during Myxococcus xanthus development

PubMed Central

Cotter, Christopher R.; Schüttler, Heinz-Bernd; Igoshin, Oleg A.; Shimkets, Lawrence J.

2017-01-01

Collective cell movement is critical to the emergent properties of many multicellular systems, including microbial self-organization in biofilms, embryogenesis, wound healing, and cancer metastasis. However, even the best-studied systems lack a complete picture of how diverse physical and chemical cues act upon individual cells to ensure coordinated multicellular behavior. Known for its social developmental cycle, the bacterium Myxococcus xanthus uses coordinated movement to generate three-dimensional aggregates called fruiting bodies. Despite extensive progress in identifying genes controlling fruiting body development, cell behaviors and cell–cell communication mechanisms that mediate aggregation are largely unknown. We developed an approach to examine emergent behaviors that couples fluorescent cell tracking with data-driven models. A unique feature of this approach is the ability to identify cell behaviors affecting the observed aggregation dynamics without full knowledge of the underlying biological mechanisms. The fluorescent cell tracking revealed large deviations in the behavior of individual cells. Our modeling method indicated that decreased cell motility inside the aggregates, a biased walk toward aggregate centroids, and alignment among neighboring cells in a radial direction to the nearest aggregate are behaviors that enhance aggregation dynamics. Our modeling method also revealed that aggregation is generally robust to perturbations in these behaviors and identified possible compensatory mechanisms. The resulting approach of directly combining behavior quantification with data-driven simulations can be applied to more complex systems of collective cell movement without prior knowledge of the cellular machinery and behavioral cues. PMID:28533367

A Two-Dimensional Numerical Investigation of Transport of Malaria-Infected Red Blood Cells in Stenotic Microchannels

PubMed Central

Tao, Yong; Rongin, Uwitije; Xing, Zhongwen

2016-01-01

The malaria-infected red blood cells experience a significant decrease in cell deformability and increase in cell membrane adhesion. Blood hemodynamics in microvessels is significantly affected by the alteration of the mechanical property as well as the aggregation of parasitized red blood cells. In this study, we aim to numerically study the connection between cell-level mechanobiological properties of human red blood cells and related malaria disease state by investigating the transport of multiple red blood cell aggregates passing through microchannels with symmetric stenosis. Effects of stenosis magnitude, aggregation strength, and cell deformability on cell rheology and flow characteristics were studied by a two-dimensional model using the fictitious domain-immersed boundary method. The results indicated that the motion and dissociation of red blood cell aggregates were influenced by these factors and the flow resistance increases with the increase of aggregating strength and cell stiffness. Further, the roughness of the velocity profile was enhanced by cell aggregation, which considerably affected the blood flow characteristics. The study may assist us in understanding cellular-level mechanisms in disease development. PMID:28105411

Self-organized, near-critical behavior during aggregation in Dictyostelium discoideum

NASA Astrophysics Data System (ADS)

de Palo, Giovanna; Yi, Darvin; Gregor, Thomas; Endres, Robert

During starvation, the social amoeba Dictyostelium discoideum aggregates artfully via pattern formation into a multicellular slug and finally spores. The aggregation process is mediated by the secretion and sensing of cyclic adenosine monophosphate, leading to the synchronized movement of cells. The whole process is a remarkable example of collective behavior, spontaneously emerging from single-cell chemotaxis. Despite this phenomenon being broadly studied, a precise characterization of the transition from single cells to multicellularity has been elusive. Here, using fluorescence imaging data of thousands of cells, we investigate the role of cell shape in aggregation, demonstrating remarkable transitions in cell behavior. To better understand their functional role, we analyze cell-cell correlations and provide evidence for self-organization at the onset of aggregation (as opposed to leader cells), with features of criticality in this finite system. To capture the mechanism of self-organization, we extend a detailed single-cell model of D.discoideum chemotaxis by adding cell-cell communication. We then use these results to extract a minimal set of rules leading to aggregation in the population model. If universal, similar rules may explain other types of collective cell behavior.

Controlled Assembly of Biocompatible Metallic Nanoaggregates Using a Small Molecule Crosslinker

PubMed Central

Van Haute, Desiree; Longmate, Julia M.; Berlin, Jacob M.

2015-01-01

By introducing a capping step and controlling reaction parameters, the assembly of metallic nanoparticle aggregates can be achieved using a small molecule crosslinker. Aggregates can be assembled from particles of varied size and composition and the size of the aggregates can be systematically adjusted. Following cell uptake of 60 nm aggregates, the aggregates are stable and non-toxic to macrophage cells up to 55mM Au. PMID:26208123

Modulation of invasive phenotype by interstitial pressure-driven convection in aggregates of human breast cancer cells.

PubMed

Tien, Joe; Truslow, James G; Nelson, Celeste M

2012-01-01

This paper reports the effect of elevated pressure on the invasive phenotype of patterned three-dimensional (3D) aggregates of MDA-MB-231 human breast cancer cells. We found that the directionality of the interstitial pressure profile altered the frequency of invasion by cells located at the surface of an aggregate. In particular, application of pressure at one end of an aggregate suppressed invasion at the opposite end. Experimental alteration of the configuration of cell aggregates and computational modeling of the resulting flow and solute concentration profiles revealed that elevated pressure inhibited invasion by altering the chemical composition of the interstitial fluid near the surface of the aggregate. Our data reveal a link between hydrostatic pressure, interstitial convection, and invasion.

Division of labour and the evolution of multicellularity

PubMed Central

Ispolatov, Iaroslav; Ackermann, Martin; Doebeli, Michael

2012-01-01

Understanding the emergence and evolution of multicellularity and cellular differentiation is a core problem in biology. We develop a quantitative model that shows that a multicellular form emerges from genetically identical unicellular ancestors when the compartmentalization of poorly compatible physiological processes into component cells of an aggregate produces a fitness advantage. This division of labour between the cells in the aggregate occurs spontaneously at the regulatory level owing to mechanisms present in unicellular ancestors and does not require any genetic predisposition for a particular role in the aggregate or any orchestrated cooperative behaviour of aggregate cells. Mathematically, aggregation implies an increase in the dimensionality of phenotype space that generates a fitness landscape with new fitness maxima, in which the unicellular states of optimized metabolism become fitness saddle points. Evolution of multicellularity is modelled as evolution of a hereditary parameter: the propensity of cells to stick together, which determines the fraction of time a cell spends in the aggregate form. Stickiness can increase evolutionarily owing to the fitness advantage generated by the division of labour between cells in an aggregate. PMID:22158952

Real-time monitoring of quorum sensing in 3D-printed bacterial aggregates using scanning electrochemical microscopy.

PubMed

Connell, Jodi L; Kim, Jiyeon; Shear, Jason B; Bard, Allen J; Whiteley, Marvin

2014-12-23

Microbes frequently live in nature as small, densely packed aggregates containing ∼10(1)-10(5) cells. These aggregates not only display distinct phenotypes, including resistance to antibiotics, but also, serve as building blocks for larger biofilm communities. Aggregates within these larger communities display nonrandom spatial organization, and recent evidence indicates that this spatial organization is critical for fitness. Studying single aggregates as well as spatially organized aggregates remains challenging because of the technical difficulties associated with manipulating small populations. Micro-3D printing is a lithographic technique capable of creating aggregates in situ by printing protein-based walls around individual cells or small populations. This 3D-printing strategy can organize bacteria in complex arrangements to investigate how spatial and environmental parameters influence social behaviors. Here, we combined micro-3D printing and scanning electrochemical microscopy (SECM) to probe quorum sensing (QS)-mediated communication in the bacterium Pseudomonas aeruginosa. Our results reveal that QS-dependent behaviors are observed within aggregates as small as 500 cells; however, aggregates larger than 2,000 bacteria are required to stimulate QS in neighboring aggregates positioned 8 μm away. These studies provide a powerful system to analyze the impact of spatial organization and aggregate size on microbial behaviors.

Molecular mechanisms of platelet activation and aggregation induced by breast cancer cells.

PubMed

Zarà, Marta; Canobbio, Ilaria; Visconte, Caterina; Canino, Jessica; Torti, Mauro; Guidetti, Gianni Francesco

2018-08-01

Tumor cell-induced platelet aggregation represents a critical process both for successful metastatic spread of the tumor and for the development of thrombotic complications in cancer patients. To get further insights into this process, we investigated and compared the molecular mechanisms of platelet aggregation induced by two different breast cancer cell lines (MDA-MB-231 and MCF7) and a colorectal cancer cell line (Caco-2). All the three types of cancer cells were able to induce comparable platelet aggregation, which, however, was observed exclusively in the presence of CaCl 2 and autologous plasma. Aggregation was supported both by fibrinogen binding to integrin αIIbβ3 as well as by fibrin formation, and was completely prevented by the serine protease inhibitor PPACK. Platelet aggregation was preceded by generation of low amounts of thrombin, possibly through tumor cells-expressed tissue factor, and was supported by platelet activation, as revealed by stimulation of phospholipase C, intracellular Ca 2+ increase and activation of Rap1b GTPase. Pharmacological inhibition of phospholipase C, but not of phosphatidylinositol 3-kinase or Src family kinases prevented tumor cell-induced platelet aggregation. Tumor cells also induced dense granule secretion, and the stimulation of the P2Y12 receptor by released ADP was found to be necessary for complete platelet aggregation. By contrast, prevention of thromboxane A 2 synthesis by aspirin did not alter the ability of all the cancer cell lines analyzed to induce platelet aggregation. These results indicate that tumor cell-induced platelet aggregation is not related to the type of the cancer cells or to their metastatic potential, and is triggered by platelet activation and secretion driven by the generation of small amount of thrombin from plasma and supported by the positive feedback signaling through secreted ADP. Copyright © 2018 Elsevier Inc. All rights reserved.

Development of long-term primary cell aggregates from Mediterranean octocorals.

PubMed

Huete-Stauffer, Carla; Valisano, Laura; Gaino, Elda; Vezzulli, Luigi; Cerrano, Carlo

2015-09-01

In lower metazoans, the aggregative properties of dissociated cells leading to in vitro stable multicellular aggregates have furnished a remarkable experimental material to carry out investigations in various research fields. One of the main expectations is to find good models for the study in vitro of the first steps of biomineralization processes. In this study, we examined five common Mediterranean gorgonians (Paramuricea clavata, Corallium rubrum, Eunicella singularis, Eunicella cavolinii, and Eunicella verrucosa) using mechanical cell aggregate production techniques. In particular, we investigated the conditions of aggregate formation, their number and survival in experimental conditions, the DNA synthesizing activity using 5'-bromo-2'-deoxyuridine (BrdU) tests, and the response to calcein addition and observed the secretion of newly formed sclerites. The BrdU tests showed that cell proliferation depends on the size of aggregates and on the presence/absence of symbiotic zooxanthellae. With epifluorescent and confocal imaging from calcein addition assays, we observed the presence of calcium ions within cells, a possible clue for prediction of sclerite formation or calcium deposition. The species-specific efficiency in production of cell aggregates is correlated to the size of polyps, showing that the higher density of polyps and their diameter correspond to higher production of cell aggregates. Regarding the long-term maintenance, we obtained the best results from E. singularis, which formed multicellular aggregates of 0.245 mm ± 0.086 mm in size and maintained symbiotic association with zooxanthellae throughout the experimental run. Formation of sclerites within aggregates opens a wide field of investigation on biomineralization, since de novo sclerites were observed around 30 d after the beginning of the experiment.

Engineering a fibrocartilage spectrum through modulation of aggregate redifferentiation.

PubMed

Murphy, Meghan K; Masters, Taylor E; Hu, Jerry C; Athanasiou, Kyriacos A

2015-01-01

Expanded costochondral cells provide a clinically relevant cell source for engineering both fibrous and hyaline articular cartilage. Expanding chondrocytes in a monolayer results in a shift toward a proliferative, fibroblastic phenotype. Three-dimensional aggregate culture may, however, be used to recover chondrogenic matrix production. This study sought to engineer a spectrum of fibrous to hyaline neocartilage from a single cell source by varying the duration of three-dimensional culture following expansion. In third passage porcine costochondral cells, the effects of aggregate culture duration were assessed after 0, 8, 11, 14, and 21 days of aggregate culture and after 4 subsequent weeks of neocartilage formation. Varying the duration of aggregate redifferentiation generated a spectrum of fibrous to hyaline neocartilage. Within 8 days of aggregation, proliferation ceased, and collagen and glycosaminoglycan production increased, compared with monolayer cells. In self-assembled neocartilage, type II-to-I collagen ratio increased with increasing aggregate duration, yet glycosaminoglycan content varied minimally. Notably, 14 days of aggregate redifferentiation increased collagen content by 25%, tensile modulus by over 110%, and compressive moduli by over 50%, compared with tissue formed in the absence of redifferentiation. A spectrum of fibrous to hyaline cartilage was generated using a single, clinically relevant cell source, improving the translational potential of engineered cartilage.

Engineering a Fibrocartilage Spectrum Through Modulation of Aggregate Redifferentiation

PubMed Central

Murphy, Meghan K.; Masters, Taylor E.; Hu, Jerry C.; Athanasiou, Kyriacos A.

2015-01-01

Expanded costochondral cells provide a clinically relevant cell source for engineering both fibrous and hyaline articular cartilage. Expanding chondrocytes in monolayer results in a shift toward a proliferative, fibroblastic phenotype. Three-dimensional aggregate culture may, however, be used to recover chondrogenic matrix production. This study sought to engineer a spectrum of fibrous to hyaline neocartilage from a single cell source by varying the duration of three-dimensional culture following expansion. In third passage porcine costochondral cells, the effects of aggregate culture duration were assessed after 0, 8, 11, 14, and 21 days of aggregate culture and after 4 subsequent weeks of neocartilage formation. Varying the duration of aggregate redifferentiation generated a spectrum of fibrous to hyaline neocartilage. Within 8 days of aggregation, proliferation ceased, and collagen and glycosaminoglycan production increased, compared with monolayer cells. In self-assembled neocartilage, type II to I collagen ratio increased with increasing aggregate duration, yet glycosaminoglycan content varied minimally. Notably, 14 days of aggregate redifferentiation increased collagen content by 25%, tensile modulus by over 110%, and compressive moduli by over 50%, compared with tissue formed in the absence of redifferentiation. A spectrum of fibrous to hyaline cartilage was generated using a single, clinically relevant cell source, improving the translational potential of engineered cartilage. PMID:24380383

Optical Measurement of Cell Colonization Patterns on Individual Suspended Sediment Aggregates

NASA Astrophysics Data System (ADS)

Nguyen, Thu Ha; Tang, Fiona H. M.; Maggi, Federico

2017-10-01

Microbial processes can make substantial differences to the way in which particles settle in aquatic environments. A novel method (OMCEC, optical measurement of cell colonization) is introduced to systematically map the biological spatial distribution over individual suspended sediment aggregates settling through a water column. OMCEC was used to investigate (1) whether a carbon source concentration has an impact on cell colonization, (2) how cells colonize minerals, and (3) if a correlation between colonization patterns and aggregate geometry exists. Incubations of Saccharomyces cerevisiae and stained montmorillonite at four sucrose concentrations were tested in a settling column equipped with a full-color microparticle image velocimetry system. The acquired high-resolution images were processed to map the cell distribution on aggregates based on emission spectra separation. The likelihood of cells colonizing minerals increased with increasing sucrose concentration. Colonization patterns were classified into (i) scattered, (ii) well touched, and (iii) poorly touched, with the second being predominant. Cell clusters in well-touched patterns were found to have lower capacity dimension than those in other patterns, while the capacity dimension of the corresponding aggregates was relatively high. A strong correlation of colonization patterns with aggregate biomass fraction and properties suggests dynamic colonization mechanisms from cell attachment to minerals, to joining of isolated cell clusters, and finally cell growth over the entire aggregate. This paper introduces a widely applicable method for analyses of microbial-affected sediment dynamics and highlights the microbial control on aggregate geometry, which can improve the prediction of large-scale morphodynamics processes.

A facile in vitro model to study rapid mineralization in bone tissues.

PubMed

Deegan, Anthony J; Aydin, Halil M; Hu, Bin; Konduru, Sandeep; Kuiper, Jan Herman; Yang, Ying

2014-09-16

Mineralization in bone tissue involves stepwise cell-cell and cell-ECM interaction. Regulation of osteoblast culture microenvironments can tailor osteoblast proliferation and mineralization rate, and the quality and/or quantity of the final calcified tissue. An in vitro model to investigate the influencing factors is highly required. We developed a facile in vitro model in which an osteoblast cell line and aggregate culture (through the modification of culture well surfaces) were used to mimic intramembranous bone mineralization. The effect of culture environments including culture duration (up to 72 hours for rapid mineralization study) and aggregates size (monolayer culture as control) on mineralization rate and mineral quantity/quality were examined by osteogenic gene expression (PCR) and mineral markers (histological staining, SEM-EDX and micro-CT). Two size aggregates (on average, large aggregates were 745 μm and small 79 μm) were obtained by the facile technique with high yield. Cells in aggregate culture generated visible and quantifiable mineralized matrix within 24 hours, whereas cells in monolayer failed to do so by 72 hours. The gene expression of important ECM molecules for bone formation including collagen type I, alkaline phosphatase, osteopontin and osteocalcin, varied temporally, differed between monolayer and aggregate cultures, and depended on aggregate size. Monolayer specimens stayed in a proliferation phase for the first 24 hours, and remained in matrix synthesis up to 72 hours; whereas the small aggregates were in the maturation phase for the first 24 and 48 hour cultures and then jumped to a mineralization phase at 72 hours. Large aggregates were in a mineralization phase at all these three time points and produced 36% larger bone nodules with a higher calcium content than those in the small aggregates after just 72 hours in culture. This study confirms that aggregate culture is sufficient to induce rapid mineralization and that aggregate size determines the mineralization rate. Mineral content depended on aggregate size and culture duration. Thus, our culture system may provide a good model to study regulation factors at different development phases of the osteoblastic lineage.

Aeromonas species exhibit aggregative adherence to HEp-2 cells.

PubMed Central

Neves, M S; Nunes, M P; Milhomem, A M

1994-01-01

Clinical and environmental isolates of Aeromonas species (five A. hydrophila isolates, three A. caviae isolates, and two A. sobria isolates) were tested for their adherence to HEp-2 cells. Clinical isolates of A. hydrophila and A. sobria exhibited aggregative adherence similar to that presented by enteroadherent-aggregative Escherichia coli. Bacterial aggregates adhered to cells with a typical "stacked-brick" appearance. In contrast, A. caviae strains showed a diffuse adherence pattern. Images PMID:8027331

Red blood cell generation by three-dimensional aggregate cultivation of late erythroblasts.

PubMed

Lee, EunMi; Han, So Yeon; Choi, Hye Sook; Chun, Bokhwan; Hwang, Byunghee; Baek, Eun Jung

2015-02-01

Stem cell-derived erythroid cells hold great potential for the treatment of blood-loss anemia and for erythropoiesis research; however, cultures using conventional flat plates or bioreactors have failed to show promising results. By mimicking the in vivo bone marrow (BM) environment in which most erythroid cells are physically aggregated, we show that a three-dimensional (3D) aggregate culture system facilitates erythroid cell maturation and red blood cell (RBC) production more effectively than two-dimensional high-density cell cultivation. Late erythroblasts (polychromatic or orthochromatic erythroblasts) were differentiated from cord blood CD34(+) cells over 15 days and then allowed to form tight aggregates at a minimum density of 1×10(7) cells/mL for 2-3 days. To scale up the cell culture and to make the media supply efficient throughout the cell aggregates, several macroporous microcarriers and porous scaffolds were applied to the 3D culture system. In comparison to control culture conditions, erythroid cells in 3D aggregates were significantly more differentiated toward RBCs with significantly reduced nuclear dysplasia. When 3D culture was performed inside macroporous microcarriers, the cell culture scale was increased and cells exhibited enhanced differentiation and enucleation. Microcarriers with a pore diameter of approximately 400 μm produced more mature cells than those with a smaller pore diameter. In addition, this aggregate culture method minimized the culture space and media volume required. In conclusion, a 3D aggregate culture system can be used to generate transfusable human erythrocytes at the terminal maturation stage, mimicking the in vivo BM microenvironment. Porous structures can efficiently maximize the culture scale, enabling large-scale production of RBCs. These results enhance our understanding of the importance of physical contact among late erythroblasts for their final maturation into RBCs.

Isolation and characterization of the hemichrome-stabilized membrane protein aggregates from sickle erythrocytes. Major site of autologous antibody binding.

PubMed

Kannan, R; Labotka, R; Low, P S

1988-09-25

Because the interaction of denatured hemoglobins (i.e. hemichromes) with the red cell membrane has been associated with several abnormalities commonly observed in hemichrome-containing erythrocytes, we have undertaken to isolate and characterize the hemichrome-rich membrane protein aggregates from sickle cells. The aggregates were isolated by two procedures: one at low ionic strength by centrifugation of detergent-solubilized spectrin-depleted inside-out vesicles, and the other at physiological ionic strength by detergent solubilization of whole cells followed by cytoskeletal disruption and centrifugation. The extensively washed aggregates obtained by both methods yielded similar results. These insoluble complexes were found to be highly cross-linked by predominantly intermolecular disulfide bonds; however, other nonreducible covalent linkages were also observed. Both in the presence and absence of reducing agents, the aggregate disintegrated when the hemichromes were removed by high ionic strength, suggesting that the aggregate depended heavily on the cohesive properties of the hemichromes for stability. Protein assays demonstrated that the aggregates comprised approximately 1.3% of the total membrane protein, roughly two-thirds of which appeared to be globin chains. Other major components identified in the aggregate were band 3, ankyrin, bands 4.1, 4.9, and 5, glycophorins A and B, and autologous IgG. Quantitative analysis of the IgG content demonstrated that three-fourths of the surface-bound IgG on washed sickle cells was clustered at these aggregate sites, representing an enrichment of approximately 250-fold over nonaggregated regions of the membrane. Since clustered cell surface IgG is thought to trigger removal of erythrocytes from circulation, the hemichrome-induced membrane reorganization at these aggregate sites may be an important cause of the greatly shortened life span of sickle cells.

Cell culture media impact on drug product solution stability.

PubMed

Purdie, Jennifer L; Kowle, Ronald L; Langland, Amie L; Patel, Chetan N; Ouyang, Anli; Olson, Donald J

2016-07-08

To enable subcutaneous administration of monoclonal antibodies, drug product solutions are often needed at high concentrations. A significant risk associated with high drug product concentrations is an increase in aggregate level over the shelf-life dating period. While much work has been done to understand the impact of drug product formulation on aggregation, there is limited understanding of the link between cell culture process conditions and soluble aggregate growth in drug product. During cell culture process development, soluble aggregates are often measured at harvest using cell-free material purified by Protein A chromatography. In the work reported here, cell culture media components were evaluated with respect to their impact on aggregate levels in high concentration solution drug product during accelerated stability studies. Two components, cysteine and ferric ammonium citrate, were found to impact aggregate growth rates in our current media (version 1) leading to the development of new chemically defined media and concentrated feed formulations. The new version of media and associated concentrated feeds (version 2) were evaluated across four cell lines producing recombinant IgG4 monoclonal antibodies and a bispecific antibody. In all four cell lines, the version 2 media reduced aggregate growth over the course of a 12 week accelerated stability study compared with the version 1 media, although the degree to which aggregate growth decreased was cell line dependent. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:998-1008, 2016. © 2016 American Institute of Chemical Engineers.

Peanut flour aggregation with polyphenolic extracts derived from peanut skin inhibits IgE binding capacity and attenuates RBL-2H3 cells degranulation via MAPK signaling pathway.

PubMed

Bansode, Rishipal R; Plundrich, Nathalie J; Randolph, Priscilla D; Lila, Mary Ann; Williams, Leonard L

2018-10-15

This study investigates the anti-allergic properties of peanut skin polyphenols (PSP)-enriched peanut (PN) protein aggregates. PSP was blended with PN flour at concentrations of 5, 10, 15, 30, and 40% (w/w). Rat basophil leukemia cells (RBL-2H3) were sensitized with either anti-DNP-IgE or PN-allergic plasma followed by co-exposure to unmodified PN flour (control) or PSP-PN protein aggregates and Ca 2+ ionophore, ionomycin. Immunoblotting and staining were performed to measure the IgE binding capacity of PSP-PN aggregates. Results showed that 30% PSP-PN aggregate significantly reduced β-hexosaminidase and histamine levels by 54.2% and 49.2%, respectively compared with control. Immunoblotting results revealed 40% PSP-PN aggregates significantly decreased IgE binding by 19%. The phosphorylation of p44/42 MAPK was significantly reduced while phosphorylation of p38 MAPK and SAPK/JNK increased upon PSP-PN protein aggregate exposure to the cells. Our results show that aggregation of PSP to PN proteins reduces allergic response by inhibiting Ca 2+ -induced MAPK-dependent cell degranulation. Copyright © 2018 Elsevier Ltd. All rights reserved.

The effects of serum leptin levels on thrombocyte aggregation in peritoneal dialysis patients.

PubMed

Bakirdogen, Serkan; Eren, Necmi; Bek, Sibel Gokcay; Mehtap, Ozgur; Cekmen, Mustafa Baki

2016-01-01

Serum leptin levels of chronic kidney disease patients have been detected higher than normal population. The aim of this study was to investigate the effects of serum leptin levels on thrombocyte aggregation in peritoneal dialysis patients. Fourty three peritoneal dialysis patients were included in the study. Thrombocyte aggregation was calculated from the whole blood subsequently the effects of different concentrations of human recombinant leptin on thrombocyte aggregations were investigated. Four test cells were used for this process. While leptin was not added into the first test cell, increasing amounts of leptin was added into the second, third and fourth test cells to attain the concentrations of 25, 50 and 100 ng/ml respectively. Thrombocyte aggregation was inhibited by recombinant leptin in peritoneal dialysis patients. Thrombocyte aggregation mean values were found statistically significantly higher in first test cell when compared to leptin groups in peritoneal dialysis patients. For leptin groups we could not find any statistically significant differences for thrombocyte aggregation mean values between any of the groups. Further studies with larger number of peritoneal dialysis patients are required to prove the action of leptin on thrombocyte aggregation.

The effects of serum leptin levels on thrombocyte aggregation in peritoneal dialysis patients

PubMed Central

Bakirdogen, Serkan; Eren, Necmi; Bek, Sibel Gokcay; Mehtap, Ozgur; Cekmen, Mustafa Baki

2016-01-01

Objective: Serum leptin levels of chronic kidney disease patients have been detected higher than normal population. The aim of this study was to investigate the effects of serum leptin levels on thrombocyte aggregation in peritoneal dialysis patients. Methods: Fourty three peritoneal dialysis patients were included in the study. Thrombocyte aggregation was calculated from the whole blood subsequently the effects of different concentrations of human recombinant leptin on thrombocyte aggregations were investigated. Four test cells were used for this process. While leptin was not added into the first test cell, increasing amounts of leptin was added into the second, third and fourth test cells to attain the concentrations of 25, 50 and 100 ng/ml respectively. Results: Thrombocyte aggregation was inhibited by recombinant leptin in peritoneal dialysis patients. Thrombocyte aggregation mean values were found statistically significantly higher in first test cell when compared to leptin groups in peritoneal dialysis patients. For leptin groups we could not find any statistically significant differences for thrombocyte aggregation mean values between any of the groups. Conclusion: Further studies with larger number of peritoneal dialysis patients are required to prove the action of leptin on thrombocyte aggregation. PMID:28083046

Freezing Responses in DMSO-Based Cryopreservation of Human iPS Cells: Aggregates Versus Single Cells.

PubMed

Li, Rui; Yu, Guanglin; Azarin, Samira M; Hubel, Allison

2018-05-01

Inadequate preservation methods of human induced pluripotent stem cells (hiPSCs) have impeded efficient reestablishment of cell culture after the freeze-thaw process. In this study, we examined roles of the cooling rate, seeding temperature, and difference between cell aggregates (3-50 cells) and single cells in controlled rate freezing of hiPSCs. Intracellular ice formation (IIF), post-thaw membrane integrity, cell attachment, apoptosis, and cytoskeleton organization were evaluated to understand the different freezing responses between hiPSC single cells and aggregates, among cooling rates of 1, 3, and 10°C/min, and between seeding temperatures of -4°C and -8°C. Raman spectroscopy images of ice showed that a lower seeding temperature (-8°C) did not affect IIF in single cells, but significantly increased IIF in aggregates, suggesting higher sensitivity of aggregates to supercooling. In the absence of IIF, Raman images showed greater variation of dimethyl sulfoxide concentration across aggregates than single cells, suggesting cryoprotectant transport limitations in aggregates. The ability of cryopreserved aggregates to attach to culture substrates did not correlate with membrane integrity for the wide range of freezing parameters, indicating inadequacy of using only membrane integrity-based optimization metrics. Lower cooling rates (1 and 3°C/min) combined with higher seeding temperature (-4°C) were better at preventing IIF and preserving cell function than a higher cooling rate (10°C/min) or lower seeding temperature (-8°C), proving the seeding temperature range of -7°C to -12°C from literature to be suboptimal. Unique f-actin cytoskeletal organization into a honeycomb-like pattern was observed in postpassage and post-thaw colonies and correlated with successful reestablishment of cell culture.

Formation and spreading of TDP-43 aggregates in cultured neuronal and glial cells demonstrated by time-lapse imaging

PubMed Central

Ishii, Tomohiro; Kawakami, Emiko; Endo, Kentaro; Misawa, Hidemi; Watabe, Kazuhiko

2017-01-01

TAR DNA-binding protein 43 (TDP-43) is a main constituent of cytoplasmic aggregates in neuronal and glial cells in cases of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. We have previously demonstrated that adenovirus-transduced artificial TDP-43 cytoplasmic aggregates formation is enhanced by proteasome inhibition in vitro and in vivo. However, the relationship between cytoplasmic aggregate formation and cell death remains unclear. In the present study, rat neural stem cell lines stably transfected with EGFP- or Sirius-expression vectors under the control of tubulin beta III, glial fibrillary acidic protein, or 2′,3′-cyclic nucleotide 3′-phosphodiesterase promoter were differentiated into neurons, astrocytes, and oligodendrocytes, respectively, in the presence of retinoic acid. The differentiated cells were then transduced with adenoviruses expressing DsRed-tagged human wild type and C-terminal fragment TDP-43 under the condition of proteasome inhibition. Time-lapse imaging analyses revealed growing cytoplasmic aggregates in the transduced neuronal and glial cells, followed by collapse of the cell. The aggregates remained insoluble in culture media, consisted of sarkosyl-insoluble granular materials, and contained phosphorylated TDP-43. Moreover, the released aggregates were incorporated into neighboring neuronal cells, suggesting cell-to-cell spreading. The present study provides a novel tool for analyzing the detailed molecular mechanisms of TDP-43 proteinopathy in vitro. PMID:28599005

Diatom-associated bacteria are required for aggregation of Thalassiosira weissflogii

PubMed Central

Gärdes, Astrid; Iversen, Morten H; Grossart, Hans-Peter; Passow, Uta; Ullrich, Matthias S

2011-01-01

Aggregation of algae, mainly diatoms, is an important process in marine systems leading to the settling of particulate organic carbon predominantly in the form of marine snow. Exudation products of phytoplankton form transparent exopolymer particles (TEP), which acts as the glue for particle aggregation. Heterotrophic bacteria interacting with phytoplankton may influence TEP formation and phytoplankton aggregation. This bacterial impact has not been explored in detail. We hypothesized that bacteria attaching to Thalassiosira weissflogii might interact in a yet-to-be determined manner, which could impact TEP formation and aggregate abundance. The role of individual T. weissflogii-attaching and free-living new bacterial isolates for TEP production and diatom aggregation was investigated in vitro. T. weissflogii did not aggregate in axenic culture, and striking differences in aggregation dynamics and TEP abundance were observed when diatom cultures were inoculated with either diatom-attaching or free-living bacteria. The data indicated that free-living bacteria might not influence aggregation whereas bacteria attaching to diatom cells may increase aggregate formation. Interestingly, photosynthetically inactivated T. weissflogii cells did not aggregate regardless of the presence of bacteria. Comparison of aggregate formation, TEP production, aggregate sinking velocity and solid hydrated density revealed remarkable differences. Both, photosynthetically active T. weissflogii and specific diatom-attaching bacteria were required for aggregation. It was concluded that interactions between heterotrophic bacteria and diatoms increased aggregate formation and particle sinking and thus may enhance the efficiency of the biological pump. PMID:20827289

Study of the cell activity in three-dimensional cell culture by using Raman spectroscopy

NASA Astrophysics Data System (ADS)

Arunngam, Pakajiraporn; Mahardika, Anggara; Hiroko, Matsuyoshi; Andriana, Bibin Bintang; Tabata, Yasuhiko; Sato, Hidetoshi

2018-02-01

The purpose of this study is to develop a estimation technique of local cell activity in cultured 3D cell aggregate with gelatin hydrogel microspheres by using Raman spectroscopy. It is an invaluable technique allowing real-time, nondestructive, and invasive measurement. Cells in body generally exist in 3D structure, which physiological cell-cell interaction enhances cell survival and biological functions. Although a 3D cell aggregate is a good model of the cells in living tissues, it was difficult to estimate their physiological conditions because there is no effective technique to make observation of intact cells in the 3D structure. In this study, cell aggregates were formed by MC3T-E1 (pre-osteoblast) cells and gelatin hydrogel microspheres. In appropriate condition MC3T-E1 cells can differentiate into osteoblast. We assume that the activity of the cell would be different according to the location in the aggregate because the cells near the surface of the aggregate have more access to oxygen and nutrient. Raman imaging technique was applied to measure 3D image of the aggregate. The concentration of the hydroxyapatite (HA) is generated by osteoblast was estimated with a strong band at 950-970 cm-1 which assigned to PO43- in HA. It reflects an activity of the specific site in the cell aggregate. The cell density in this specific site was analyzed by multivariate analysis of the 3D Raman image. Hence, the ratio between intensity and cell density in the site represents the cell activity.

Aggregate formation affects ultrasonic disruption of microalgal cells.

PubMed

Wang, Wei; Lee, Duu-Jong; Lai, Juin-Yih

2015-12-01

Ultrasonication is a cell disruption process of low energy efficiency. This study dosed K(+), Ca(2+) and Al(3+) to Chlorella vulgaris cultured in Bold's Basal Medium at 25°C and measured the degree of cell disruption under ultrasonication. Adding these metal ions yielded less negatively charged surfaces of cells, while with the latter two ions large and compact cell aggregates were formed. The degree of cell disruption followed: control=K(+)>Ca(2+)>Al(3+) samples. Surface charges of cells and microbubbles have minimal effects on the microbubble number in the proximity of the microalgal cells. Conversely, cell aggregates with large size and compact interior resist cell disruption under ultrasonication. Staining tests revealed high diffusional resistance of stains over the aggregate interior. Microbubbles may not be effective generated and collapsed inside the compact aggregates, hence leading to low cell disruption efficiencies. Effective coagulation/flocculation in cell harvesting may lead to adverse effect on subsequent cell disruption efficiency. Copyright © 2015 Elsevier Ltd. All rights reserved.

Using Human iPSC-Derived Neurons to Model TAU Aggregation

PubMed Central

Verheyen, An; Diels, Annick; Dijkmans, Joyce; Oyelami, Tutu; Meneghello, Giulia; Mertens, Liesbeth; Versweyveld, Sofie; Borgers, Marianne; Buist, Arjan; Peeters, Pieter; Cik, Miroslav

2015-01-01

Alzheimer’s disease and frontotemporal dementia are amongst the most common forms of dementia characterized by the formation and deposition of abnormal TAU in the brain. In order to develop a translational human TAU aggregation model suitable for screening, we transduced TAU harboring the pro-aggregating P301L mutation into control hiPSC-derived neural progenitor cells followed by differentiation into cortical neurons. TAU aggregation and phosphorylation was quantified using AlphaLISA technology. Although no spontaneous aggregation was observed upon expressing TAU-P301L in neurons, seeding with preformed aggregates consisting of the TAU-microtubule binding repeat domain triggered robust TAU aggregation and hyperphosphorylation already after 2 weeks, without affecting general cell health. To validate our model, activity of two autophagy inducers was tested. Both rapamycin and trehalose significantly reduced TAU aggregation levels suggesting that iPSC-derived neurons allow for the generation of a biologically relevant human Tauopathy model, highly suitable to screen for compounds that modulate TAU aggregation. PMID:26720731

Amyloid-like aggregation of provasopressin in diabetes insipidus and secretory granule sorting.

PubMed

Beuret, Nicole; Hasler, Franziska; Prescianotto-Baschong, Cristina; Birk, Julia; Rutishauser, Jonas; Spiess, Martin

2017-01-26

Aggregation of peptide hormone precursors in the trans-Golgi network is an essential process in the biogenesis of secretory granules in endocrine cells. It has recently been proposed that this aggregation corresponds to the formation of functional amyloids. Our previous finding that dominant mutations in provasopressin, which cause cell degeneration and diabetes insipidus, prevent native folding and produce fibrillar aggregates in the endoplasmic reticulum (ER) might thus reflect mislocalized amyloid formation by sequences that evolved to mediate granule sorting. Here we identified two sequences responsible for fibrillar aggregation of mutant precursors in the ER: the N-terminal vasopressin nonapeptide and the C-terminal glycopeptide. To test their role in granule sorting, the glycopeptide was deleted and/or vasopressin mutated to inactivate ER aggregation while still permitting precursor folding and ER exit. These mutations strongly reduced sorting into granules and regulated secretion in endocrine AtT20 cells. The same sequences - vasopressin and the glycopeptide - mediate physiological aggregation of the wild-type hormone precursor into secretory granules and the pathological fibrillar aggregation of disease mutants in the ER. These findings support the amyloid hypothesis for secretory granule biogenesis.

Internalization of aggregated photosensitizers by tumor cells: subcellular time-resolved fluorescence spectroscopy on derivatives of pyropheophorbide-a ethers and chlorin e6 under femtosecond one- and two-photon excitations.

PubMed

Kelbauskas, L; Dietel, W

2002-12-01

Amphiphilic sensitizers self-associate in aqueous environments and form aggregated species that exhibit no or only negligible photodynamic activity. However, amphiphilic photosensitizers number among the most potent agents of photodynamic therapy. The processes by which these sensitizers are internalized into tumor cells have yet to be fully elucidated and thus remain the subject of debate. In this study the uptake of photosensitizer aggregates into tumor cells was examined directly using subcellular time-resolved fluorescence spectroscopy with a high temporal resolution (20-30 ps) and high sensitivity (time-correlated single-photon counting). The investigations were performed on selected sensitizers that exhibit short fluorescence decay times (< 50 ps) in aggregated form. Derivatives of pyropheophorbide-a ether and chlorin e6 with varying lipophilicity were used for the study. The characteristic fluorescence decay times and spectroscopic features of the sensitizer aggregates measured in aqueous solution also could be observed in A431 human endothelial carcinoma cells administered with these photosensitizers. This shows that tumor cells can internalize sensitizers in aggregated form. Uptake of aggregates and their monomerization inside cells were demonstrated directly for the first time by means of fluorescence lifetime imaging with a high temporal resolution. Internalization of the aggregates seems to be endocytosis mediated. The degree of their monomerization in tumor cells is strongly influenced by the lipophilicity of the compounds.

Dye-sensitized solar cell employing zinc oxide aggregates grown in the presence of lithium

DOEpatents

Zhang, Qifeng; Cao, Guozhong

2013-10-15

Provided are a novel ZnO dye-sensitized solar cell and method of fabricating the same. In one embodiment, deliberately added lithium ions are used to mediate the growth of ZnO aggregates. The use of lithium provides ZnO aggregates that have advantageous microstructure, morphology, crystallinity, and operational characteristics. Employing lithium during aggregate synthesis results in a polydisperse collection of ZnO aggregates favorable for porosity and light scattering. The resulting nanocrystallites forming the aggregates have improved crystallinity and more favorable facets for dye molecule absorption. The lithium synthesis improves the surface stability of ZnO in acidic dyes. The procedures developed and disclosed herein also help ensure the formation of an aggregate film that has a high homogeneity of thickness, a high packing density, a high specific surface area, and good electrical contact between the film and the fluorine-doped tin oxide electrode and among the aggregate particles.

A 31-residue peptide induces aggregation of tau's microtubule-binding region in cells

NASA Astrophysics Data System (ADS)

Stöhr, Jan; Wu, Haifan; Nick, Mimi; Wu, Yibing; Bhate, Manasi; Condello, Carlo; Johnson, Noah; Rodgers, Jeffrey; Lemmin, Thomas; Acharya, Srabasti; Becker, Julia; Robinson, Kathleen; Kelly, Mark J. S.; Gai, Feng; Stubbs, Gerald; Prusiner, Stanley B.; Degrado, William F.

2017-09-01

The self-propagation of misfolded conformations of tau underlies neurodegenerative diseases, including Alzheimer's. There is considerable interest in discovering the minimal sequence and active conformational nucleus that defines this self-propagating event. The microtubule-binding region, spanning residues 244-372, reproduces much of the aggregation behaviour of tau in cells and animal models. Further dissection of the amyloid-forming region to a hexapeptide from the third microtubule-binding repeat resulted in a peptide that rapidly forms fibrils in vitro. We show that this peptide lacks the ability to seed aggregation of tau244-372 in cells. However, as the hexapeptide is gradually extended to 31 residues, the peptides aggregate more slowly and gain potent activity to induce aggregation of tau244-372 in cells. X-ray fibre diffraction, hydrogen-deuterium exchange and solid-state NMR studies map the beta-forming region to a 25-residue sequence. Thus, the nucleus for self-propagating aggregation of tau244-372 in cells is packaged in a remarkably small peptide.

The nuclear lamina promotes telomere aggregation and centromere peripheral localization during senescence of human mesenchymal stem cells.

PubMed

Raz, Vered; Vermolen, Bart J; Garini, Yuval; Onderwater, Jos J M; Mommaas-Kienhuis, Mieke A; Koster, Abraham J; Young, Ian T; Tanke, Hans; Dirks, Roeland W

2008-12-15

Ex vivo, human mesenchymal stem cells (hMSCs) undergo spontaneous cellular senescence after a limited number of cell divisions. Intranuclear structures of the nuclear lamina were formed in senescent hMSCs, which are identified by the presence of Hayflick-senescence-associated factors. Notably, spatial changes in lamina shape were observed before the Hayflick senescence-associated factors, suggesting that the lamina morphology can be used as an early marker to identify senescent cells. Here, we applied quantitative image-processing tools to study the changes in nuclear architecture during cell senescence. We found that centromeres and telomeres colocalised with lamina intranuclear structures, which resulted in a preferred peripheral distribution in senescent cells. In addition, telomere aggregates were progressively formed during cell senescence. Once formed, telomere aggregates showed colocalization with gamma-H2AX but not with TERT, suggesting that telomere aggregates are sites of DNA damage. We also show that telomere aggregation is associated with lamina intranuclear structures, and increased telomere binding to lamina proteins is found in cells expressing lamina mutants that lead to increases in lamina intranuclear structures. Moreover, three-dimensional image processing revealed spatial overlap between telomere aggregates and lamina intranuclear structures. Altogether, our data suggest a mechanical link between changes in lamina spatial organization and the formation of telomere aggregates during senescence of hMSCs, which can possibly contribute to changes in nuclear activity during cell senescence.

Bacterial and iron oxide aggregates mediate secondary iron mineral formation: green rust versus magnetite.

PubMed

Zegeye, A; Mustin, C; Jorand, F

2010-06-01

In the presence of methanoate as electron donor, Shewanella putrefaciens, a Gram-negative, facultative anaerobe, is able to transform lepidocrocite (gamma-FeOOH) to secondary Fe (II-III) minerals such as carbonated green rust (GR1) and magnetite. When bacterial cells were added to a gamma-FeOOH suspension, aggregates were produced consisting of both bacteria and gamma-FeOOH particles. Recently, we showed that the production of secondary minerals (GR1 vs. magnetite) was dependent on bacterial cell density and not only on iron reduction rates. Thus, gamma-FeOOH and S. putrefaciens aggregation pattern was suggested as the main mechanism driving mineralization. In this study, lepidocrocite bioreduction experiments, in the presence of anthraquinone disulfonate, were conducted by varying the [cell]/[lepidocrocite] ratio in order to determine whether different types of aggregate are formed, which may facilitate precipitation of GR1 as opposed to magnetite. Confocal laser scanning microscopy was used to analyze the relative cell surface area and lepidocrocite concentration within the aggregates and captured images were characterized by statistical methods for spatial data (i.e. variograms). These results suggest that the [cell]/[lepidocrocite] ratio influenced both the aggregate structure and the nature of the secondary iron mineral formed. Subsequently, a [cell]/[lepidocrocite] ratio above 1 x 10(7) cells mmol(-1) leads to densely packed aggregates and to the formation of GR1. Below this ratio, looser aggregates are formed and magnetite was systematically produced. The data presented in this study bring us closer to a more comprehensive understanding of the parameters governing the formation of minerals in dense bacterial suspensions and suggest that screening mineral-bacteria aggregate structure is critical to understanding (bio)mineralization pathways.

Normal and system lupus erythematosus red blood cell interactions studied by double trap optical tweezers: direct measurements of aggregation forces

NASA Astrophysics Data System (ADS)

Khokhlova, Maria D.; Lyubin, Eugeny V.; Zhdanov, Alexander G.; Rykova, Sophia Yu.; Sokolova, Irina A.; Fedyanin, Andrey A.

2012-02-01

Direct measurements of aggregation forces in piconewton range between two red blood cells in pair rouleau are performed under physiological conditions using double trap optical tweezers. Aggregation and disaggregation properties of healthy and pathologic (system lupus erythematosis) blood samples are analyzed. Strong difference in aggregation speed and behavior is revealed using the offered method which is proposed to be a promising tool for SLE monitoring at single cell level.

Waves and aggregation patterns in myxobacteria

NASA Astrophysics Data System (ADS)

Igoshin, Oleg A.; Welch, Roy; Kaiser, Dale; Oster, George

2004-03-01

Under starvation conditions, a population of myxobacteria aggregates to build a fruiting body whose shape is species-specific and within which the cells sporulate. Early in this process, cells often pass through a "ripple phase" characterized by traveling linear, concentric, and spiral waves. These waves are different from the waves observed during slime mold aggregation that depend on diffusible morphogens, because myxobacteria communicate by direct contact. The difference is most dramatic when waves collide: rather than annihilating one another, myxobacterial waves appear to pass through one another unchanged. Under certain conditions, the spacing and location of the nascent fruiting bodies is determined by the wavelength and pattern of the waves. Later in fruiting body development, waves are replaced by streams of cells that circulate around small initial aggregates enlarging and rounding them. Still later, pairs of motile aggregates coalesce to form larger aggregates that develop into fruiting bodies. Here we present a mathematical model that quantitatively explains these wave and aggregation phenomena.

Comparison of three optical methods to study erythrocyte aggregation.

PubMed

Zhao, H; Wang, X; Stoltz, J F

1999-01-01

The aim of this work was to evaluate three optical methods designed to determine erythrocyte aggregation: Erythroaggregometer (EA; Regulest, France), Laser-assisted Optical Rotational Cell Analyzer (LORCA; Mechatronics, Netherlands) and Fully Automatic Erythrocyte Aggregometer (FAEA; Myrenne, GmbH, Germany). Blood samples were taken from fifty donors (26 males and 24 females). The aggregation of normal red blood cell (RBC) and RBCs suspended in three normo- and hyperaggregating suspending media was studied. The results revealed some significant correlations between parameters measured by these instruments, in particular, between the indexes of aggregation of EA and LORCA. Further, RBC aggregation of multiple myeloma patients was also studied and a hyper erythrocyte aggregation state was found by EA and LORCA.

Morphological classification of bioaerosols from composting using scanning electron microscopy

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

Tamer Vestlund, A.; FIRA International Ltd., Maxwell Road, Stevenage, Herts SG1 2EW; Al-Ashaab, R.

2014-07-15

Highlights: • Bioaerosols were captured using the filter method. • Bioaerosols were analysed using scanning electron microscope. • Bioaerosols were classified on the basis of morphology. • Single small cells were found more frequently than aggregates and larger cells. • Smaller cells may disperse further than heavier aggregate structures. - Abstract: This research classifies the physical morphology (form and structure) of bioaerosols emitted from open windrow composting. Aggregation state, shape and size of the particles captured are reported alongside the implications for bioaerosol dispersal after release. Bioaerosol sampling took place at a composting facility using personal air filter samplers. Samplesmore » were analysed using scanning electron microscopy. Particles were released mainly as small (<1 μm) single, spherical cells, followed by larger (>1 μm) single cells, with aggregates occurring in smaller proportions. Most aggregates consisted of clusters of 2–3 particles as opposed to chains, and were <10 μm in size. No cells were attached to soil debris or wood particles. These small single cells or small aggregates are more likely to disperse further downwind from source, and cell viability may be reduced due to increased exposure to environmental factors.« less

Aggregate formation and suspension culture of human pluripotent stem cells and differentiated progeny.

PubMed

Hookway, Tracy A; Butts, Jessica C; Lee, Emily; Tang, Hengli; McDevitt, Todd C

2016-05-15

Culture of human pluripotent stem cells (hPSC) as in vitro multicellular aggregates has been increasingly used as a method to model early embryonic development. Three-dimensional assemblies of hPSCs facilitate interactions between cells and their microenvironment to promote morphogenesis, analogous to the multicellular organization that accompanies embryogenesis. In this paper, we describe a method for reproducibly generating and maintaining populations of homogeneous three-dimensional hPSC aggregates using forced aggregation and rotary orbital suspension culture. We propose solutions to several challenges associated with the consistent formation and extended culture of cell spheroids generated from hPSCs and their differentiated progeny. Further, we provide examples to demonstrate how aggregation can be used as a tool to select specific subpopulations of cells to create homotypic spheroids, or as a means to introduce multiple cell types to create heterotypic tissue constructs. Finally, we demonstrate that the aggregation and rotary suspension method can be used to support culture and maintenance of hPSC-derived cell populations representing each of the three germ layers, underscoring the utility of this platform for culturing many different cell types. Copyright © 2015 Elsevier Inc. All rights reserved.

Intrinsic Lens Forming Potential of Mouse Lens Epithelial versus Newt Iris Pigment Epithelial Cells in Three-Dimensional Culture

PubMed Central

Nakamura, Kenta; Tsonis, Panagiotis A.

2014-01-01

Adult newts (Notophthalmus viridescens) are capable of complete lens regeneration that is mediated through dorsal iris pigment epithelial (IPE) cells transdifferentiation. In contrast, higher vertebrates such as mice demonstrate only limited lens regeneration in the presence of an intact lens capsule with remaining lens epithelial cells. To compare the intrinsic lens regeneration potential of newt IPE versus mouse lens epithelial cells (MLE), we have established a novel culture method that uses cell aggregation before culture in growth factor-reduced Matrigel™. Dorsal newt IPE aggregates demonstrated complete lens formation within 1 to 2 weeks of Matrigel culture without basic fibroblast growth factor (bFGF) supplementation, including the establishment of a peripheral cuboidal epithelial cell layer, and the appearance of central lens fibers that were positive for αA-crystallin. In contrast, the lens-forming potential of MLE cell aggregates cultured in Matrigel was incomplete and resulted in the formation of defined-size lentoids with partial optical transparency. While the peripheral cell layers of MLE aggregates were nucleated, cells in the center of aggregates demonstrated a nonapoptotic nuclear loss over a time period of 3 weeks that was representative of lens fiber formation. Matrigel culture supplementation with bFGF resulted in higher transparent bigger-size MLE aggregates that demonstrated increased appearance of βB1-crystallin expression. Our study demonstrates that bFGF is not required for induction of newt IPE aggregate-dependent lens formation in Matrigel, while the addition of bFGF seems to be beneficial for the formation of MLE aggregate-derived lens-like structures. In conclusion, the three-dimensional aggregate culture of IPE and MLE in Matrigel allows to a higher extent than older models the indepth study of the intrinsic lens-forming potential and the corresponding identification of lentogenic factors. PMID:23672748

Modeling Multivalent Ligand-Receptor Interactions with Steric Constraints on Configurations of Cell-Surface Receptor Aggregates

PubMed Central

Monine, Michael I.; Posner, Richard G.; Savage, Paul B.; Faeder, James R.; Hlavacek, William S.

2010-01-01

Abstract We use flow cytometry to characterize equilibrium binding of a fluorophore-labeled trivalent model antigen to bivalent IgE-FcεRI complexes on RBL cells. We find that flow cytometric measurements are consistent with an equilibrium model for ligand-receptor binding in which binding sites are assumed to be equivalent and ligand-induced receptor aggregates are assumed to be acyclic. However, this model predicts extensive receptor aggregation at antigen concentrations that yield strong cellular secretory responses, which is inconsistent with the expectation that large receptor aggregates should inhibit such responses. To investigate possible explanations for this discrepancy, we evaluate four rule-based models for interaction of a trivalent ligand with a bivalent cell-surface receptor that relax simplifying assumptions of the equilibrium model. These models are simulated using a rule-based kinetic Monte Carlo approach to investigate the kinetics of ligand-induced receptor aggregation and to study how the kinetics and equilibria of ligand-receptor interaction are affected by steric constraints on receptor aggregate configurations and by the formation of cyclic receptor aggregates. The results suggest that formation of linear chains of cyclic receptor dimers may be important for generating secretory signals. Steric effects that limit receptor aggregation and transient formation of small receptor aggregates may also be important. PMID:20085718

Native-like aggregates of Factor VIII (FVIII) are immunogenic von Willebrand Factor deficient and hemophilia A mice

PubMed Central

Pisal, Dipak S.; Kosloski, Matthew P.; Middaugh, C. Russell; Bankert, Richard B.; Balu-Iyer, Sathy V.

2013-01-01

The administration of recombinant Factor VIII (FVIII) is the first line therapy for Hemophilia A (HA), but 25–35% of patients develop an inhibitory antibody response. In general, the presence of aggregates contributes to unwanted immunogenic responses against therapeutic proteins. FVIII has been shown to form both native-like and non-native aggregates. Previously, we showed that non-native aggregates of FVIII are less immunogenic compared to the native protein. Here we investigated the effect of native-like aggregates of FVIII on immunogenicity in HA and von Willebrand Factor knockout (vWF−/−) mice. Mice immunized with native-like aggregates showed significantly higher inhibitory antibody titers compared to animals that received native FVIII. Following re-stimulation in vitro with native FVIII, the activation of CD4+ T cells isolated from mice immunized with native-like aggregates is ~4 fold higher than mice immunized with the native protein. Furthermore, this is associated with increases in the secretion of pro-inflammatory cytokines IL-6 and IL-17 in the native-like aggregate treatment group. The results indicate that the native-like aggregates of FVIII are more immunogenic than native FVIII for both the B cell and T cell responses. PMID:22388918

Oxygen transport and stem cell aggregation in stirred-suspension bioreactor cultures.

PubMed

Wu, Jincheng; Rostami, Mahboubeh Rahmati; Cadavid Olaya, Diana P; Tzanakakis, Emmanuel S

2014-01-01

Stirred-suspension bioreactors are a promising modality for large-scale culture of 3D aggregates of pluripotent stem cells and their progeny. Yet, cells within these clusters experience limitations in the transfer of factors and particularly O2 which is characterized by low solubility in aqueous media. Cultured stem cells under different O2 levels may exhibit significantly different proliferation, viability and differentiation potential. Here, a transient diffusion-reaction model was built encompassing the size distribution and ultrastructural characteristics of embryonic stem cell (ESC) aggregates. The model was coupled to experimental data from bioreactor and static cultures for extracting the effective diffusivity and kinetics of consumption of O2 within mouse (mESC) and human ESC (hESC) clusters. Under agitation, mESC aggregates exhibited a higher maximum consumption rate than hESC aggregates. Moreover, the reaction-diffusion model was integrated with a population balance equation (PBE) for the temporal distribution of ESC clusters changing due to aggregation and cell proliferation. Hypoxia was found to be negligible for ESCs with a smaller radius than 100 µm but became appreciable for aggregates larger than 300 µm. The integrated model not only captured the O2 profile both in the bioreactor bulk and inside ESC aggregates but also led to the calculation of the duration that fractions of cells experience a certain range of O2 concentrations. The approach described in this study can be employed for gaining a deeper understanding of the effects of O2 on the physiology of stem cells organized in 3D structures. Such frameworks can be extended to encompass the spatial and temporal availability of nutrients and differentiation factors and facilitate the design and control of relevant bioprocesses for the production of stem cell therapeutics.

Aging, mortality, and the fast growth trade-off of Schizosaccharomyces pombe

PubMed Central

Nakaoka, Hidenori; Wakamoto, Yuichi

2017-01-01

Replicative aging has been demonstrated in asymmetrically dividing unicellular organisms, seemingly caused by unequal damage partitioning. Although asymmetric segregation and inheritance of potential aging factors also occur in symmetrically dividing species, it nevertheless remains controversial whether this results in aging. Based on large-scale single-cell lineage data obtained by time-lapse microscopy with a microfluidic device, in this report, we demonstrate the absence of replicative aging in old-pole cell lineages of Schizosaccharomyces pombe cultured under constant favorable conditions. By monitoring more than 1,500 cell lineages in 7 different culture conditions, we showed that both cell division and death rates are remarkably constant for at least 50–80 generations. Our measurements revealed that the death rate per cellular generation increases with the division rate, pointing to a physiological trade-off with fast growth under balanced growth conditions. We also observed the formation and inheritance of Hsp104-associated protein aggregates, which are a potential aging factor in old-pole cell lineages, and found that these aggregates exhibited a tendency to preferentially remain at the old poles for several generations. However, the aggregates were eventually segregated from old-pole cells upon cell division and probabilistically allocated to new-pole cells. We found that cell deaths were typically preceded by sudden acceleration of protein aggregation; thus, a relatively large amount of protein aggregates existed at the very ends of the dead cell lineages. Our lineage tracking analyses, however, revealed that the quantity and inheritance of protein aggregates increased neither cellular generation time nor cell death initiation rates. Furthermore, our results demonstrated that unusually large amounts of protein aggregates induced by oxidative stress exposure did not result in aging; old-pole cells resumed normal growth upon stress removal, despite the fact that most of them inherited significant quantities of aggregates. These results collectively indicate that protein aggregates are not a major determinant of triggering cell death in S. pombe and thus cannot be an appropriate molecular marker or index for replicative aging under both favorable and stressful environmental conditions. PMID:28632741

Assessment of the ``cross-bridge''-induced interaction of red blood cells by optical trapping combined with microfluidics

NASA Astrophysics Data System (ADS)

Lee, Kisung; Wagner, Christian; Priezzhev, Alexander V.

2017-09-01

Red blood cell (RBC) aggregation is an intrinsic property of the blood that has a direct effect on the blood viscosity and circulation. Nevertheless, the mechanism behind the RBC aggregation has not been confirmed and is still under investigation with two major hypotheses, known as "depletion layer" and "cross-bridging." We aim to ultimately understand the mechanism of the RBC aggregation and clarify both models. To measure the cell interaction in vitro in different suspensions (including plasma, isotonic solution of fibrinogen, isotonic solution of fibrinogen with albumin, and phosphate buffer saline) while moving the aggregate from one solution to another, an approach combining optical trapping and microfluidics has been applied. The study reveals evidence that RBC aggregation in plasma is at least partly due to the cross-bridging mechanism. The cell interaction strength measured in the final solution was found to be significantly changed depending on the initial solution where the aggregate was formed.

The response of aggregated Pseudomonas putida CP1 cells to UV-C and UV-A/B disinfection.

PubMed

Maganha de Almeida, Ana C; Quilty, Bríd

2016-11-01

UV radiation is a spread method used worldwide for the disinfection of water. However, much of the research on the disinfection of bacterial cells by UV has focused on planktonic cells. Many bacterial cells in nature are present in clumps or aggregates, and these aggregates, which are more resistant to disinfection than their planktonic counterparts, can be problematic in engineered water systems. The current research used Pseudomonas putida (P. putida) CP1, an environmental and non-pathogenic microorganism which autoaggregates when grown under certain conditions, as a model organism to simulate aggregated cells. The study investigated the response of both the planktonic and the aggregated forms of the bacterium to UV-C (λ = 253.7 nm) and UV-A/B (λ > 300 nm) disinfection at laboratory scale in a minimal medium. The planktonic cells of P. putida CP1 were inactivated within 60 s by UV-C and in 60 min by UV-A/B; however, the aggregated cells required 120 min of UV-C treatment and 240 min of UV-A/B radiation to become inactive. The size of the aggregate was reduced following UV treatment. Although all the cells had lost culturability, viability as measured by the LIVE/DEAD ® stain and epifluorescence microscopy was not completely lost and the cells all demonstrated regrowth after overnight incubation in the dark.

Modeling the reversible kinetics of neutrophil aggregation under hydrodynamic shear.

PubMed Central

Neelamegham, S; Taylor, A D; Hellums, J D; Dembo, M; Smith, C W; Simon, S I

1997-01-01

Neutrophil emigration into inflamed tissue is mediated by beta 2-integrin and L-selectin adhesion receptors. Homotypic neutrophil aggregation is also dependent on these molecules, and it provides a model system in which to study adhesion dynamics. In the current study we formulated a mathematical model for cellular aggregation in a linear shear field based on Smoluchowski's two-body collision theory. Neutrophil suspensions activated with chemotactic stimulus and sheared in a cone-plate viscometer rapidly aggregate. Over a range of shear rates (400-800 s-1), approximately 90% of the single cells were recruited into aggregates ranging from doublets to groupings larger than sextuplets. The adhesion efficiency fit to these kinetics reached maximum levels of > 70%. Formed aggregates remained intact and resistant to shear up to 120 s, at which time they spontaneously dissociated back to singlets. The rate of cell disaggregation was linearly proportional to the applied shear rate, and it was approximately 60% lower for doublets as compared to larger aggregates. By accounting for the time-dependent changes in adhesion efficiency, disaggregation rate, and the effects of aggregate geometry, we succeeded in predicting the reversible kinetics of aggregation over a wide range of shear rates and cell concentrations. The combination of viscometry with flow cytometry and mathematical analysis as presented here represents a novel approach to differentiating between the effects of hydrodynamics and the intrinsic biological processes that control cell adhesion. Images FIGURE 3 FIGURE 5 PMID:9083659

Estimation of polydispersity in aggregating red blood cells by quantitative ultrasound backscatter analysis.

PubMed

de Monchy, Romain; Rouyer, Julien; Destrempes, François; Chayer, Boris; Cloutier, Guy; Franceschini, Emilie

2018-04-01

Quantitative ultrasound techniques based on the backscatter coefficient (BSC) have been commonly used to characterize red blood cell (RBC) aggregation. Specifically, a scattering model is fitted to measured BSC and estimated parameters can provide a meaningful description of the RBC aggregates' structure (i.e., aggregate size and compactness). In most cases, scattering models assumed monodisperse RBC aggregates. This study proposes the Effective Medium Theory combined with the polydisperse Structure Factor Model (EMTSFM) to incorporate the polydispersity of aggregate size. From the measured BSC, this model allows estimating three structural parameters: the mean radius of the aggregate size distribution, the width of the distribution, and the compactness of the aggregates. Two successive experiments were conducted: a first experiment on blood sheared in a Couette flow device coupled with an ultrasonic probe, and a second experiment, on the same blood sample, sheared in a plane-plane rheometer coupled to a light microscope. Results demonstrated that the polydisperse EMTSFM provided the best fit to the BSC data when compared to the classical monodisperse models for the higher levels of aggregation at hematocrits between 10% and 40%. Fitting the polydisperse model yielded aggregate size distributions that were consistent with direct light microscope observations at low hematocrits.

Viral Aggregation: Impact on Virus Behavior in the Environment.

PubMed

Gerba, Charles P; Betancourt, Walter Q

2017-07-05

Aggregates of viruses can have a significant impact on quantification and behavior of viruses in the environment. Viral aggregates may be formed in numerous ways. Viruses may form crystal like structures and aggregates in the host cell during replication or may form due to changes in environmental conditions after virus particles are released from the host cells. Aggregates tend to form near the isoelectric point of the virus, under the influence of certain salts and salt concentrations in solution, cationic polymers, and suspended organic matter. The given conditions under which aggregates form in the environment are highly dependent on the type of virus, type of salts in solution (cation, anion. monovalent, divalent) and pH. However, virus type greatly influences the conditions when aggregation/disaggregation will occur, making predictions difficult under any given set of water quality conditions. Most studies have shown that viral aggregates increase the survival of viruses in the environment and resistance to disinfectants, especially with more reactive disinfectants. The presence of viral aggregates may also result in overestimation of removal by filtration processes. Virus aggregation-disaggregation is a complex process and predicting the behavior of any individual virus is difficult under a given set of environmental circumstances without actual experimental data.

Biogrid--a microfluidic device for large-scale enzyme-free dissociation of stem cell aggregates.

PubMed

Wallman, Lars; Åkesson, Elisabet; Ceric, Dario; Andersson, Per Henrik; Day, Kelly; Hovatta, Outi; Falci, Scott; Laurell, Thomas; Sundström, Erik

2011-10-07

Culturing stem cells as free-floating aggregates in suspension facilitates large-scale production of cells in closed systems, for clinical use. To comply with GMP standards, the use of substances such as proteolytic enzymes should be avoided. Instead of enzymatic dissociation, the growing cell aggregates may be mechanically cut at passage, but available methods are not compatible with large-scale cell production and hence translation into the clinic becomes a severe bottle-neck. We have developed the Biogrid device, which consists of an array of micrometerscale knife edges, micro-fabricated in silicon, and a manifold in which the microgrid is placed across the central fluid channel. By connecting one side of the Biogrid to a syringe or a pump and the other side to the cell culture, the culture medium with suspended cell aggregates can be aspirated, forcing the aggregates through the microgrid, and ejected back to the cell culture container. Large aggregates are thereby dissociated into smaller fragments while small aggregates pass through the microgrid unaffected. As proof-of-concept, we demonstrate that the Biogrid device can be successfully used for repeated passage of human neural stem/progenitor cells cultured as so-called neurospheres, as well as for passage of suspension cultures of human embryonic stem cells. We also show that human neural stem/progenitor cells tolerate transient pressure changes far exceeding those that will occur in a fluidic system incorporating the Biogrid microgrids. Thus, by using the Biogrid device it is possible to mechanically passage large quantities of cells in suspension cultures in closed fluidic systems, without the use of proteolytic enzymes.

Identification of a small, naked virus in tumor-like aggregates in cell lines derived from a green turtle, Chelonia mydas, with fibropapillomas

USGS Publications Warehouse

Lu, Y.; Aguirre, A.A.; Work, Thierry M.; Balazs, G.H.; Nerurkar, V.R.; Yanagihara, R.

2000-01-01

Serial cultivation of cell lines derived from lung, testis, periorbital and tumor tissues of a green turtle (Chelonia mydas) with fibropapillomas resulted in the in vitro formation of tumor-like cell aggregates, ranging in size from 0.5 to 2.0 mm in diameter. Successful induction of tumor-like aggregates was achieved in a cell line derived from lung tissue of healthy green turtles, following inoculation with cell-free media from these tumor-bearing cell lines, suggesting the presence of a transmissible agent. Thin-section electron microscopy of the cell aggregates revealed massive collagen deposits and intranuclear naked viral particles, measuring 5095 nm in diameter. These findings, together with the morphological similarity between these tumor-like cell aggregates and the naturally occurring tumor, suggest a possible association between this novel virus and the disease. Further characterization of this small naked virus will clarify its role in etiology of green turtle fibropapilloma, a life-threatening disease of this endangered marine species.

Sequestration of synaptic proteins by alpha-synuclein aggregates leading to neurotoxicity is inhibited by small peptide

PubMed Central

Choi, Mal-Gi; Kim, Mi Jin; Kim, Do-Geun; Yu, Ri; Jang, You-Na

2018-01-01

α-Synuclein (α-syn) is a major component of Lewy bodies found in synucleinopathies including Parkinson’s disease (PD) and Dementia with Lewy Bodies (DLB). Under the pathological conditions, α-syn tends to generate a diverse form of aggregates showing toxicity to neuronal cells and able to transmit across cells. However, mechanisms by which α-syn aggregates affect cytotoxicity in neurons have not been fully elucidated. Here we report that α-syn aggregates preferentially sequester specific synaptic proteins such as vesicle-associated membrane protein 2 (VAMP2) and synaptosomal-associated protein 25 (SNAP25) through direct binding which is resistant to SDS. The sequestration effect of α-syn aggregates was shown in a cell-free system, cultured primary neurons, and PD mouse model. Furthermore, we identified a specific blocking peptide derived from VAMP2 which partially inhibited the sequestration by α-syn aggregates and contributed to reduced neurotoxicity. These results provide a mechanism of neurotoxicity mediated by α-syn aggregates and suggest that the blocking peptide interfering with the pathological role of α-syn aggregates could be useful for designing a potential therapeutic drug for the treatment of PD. PMID:29608598

Curcumin inhibits aggregation of alpha-synuclein.

PubMed

Pandey, Neeraj; Strider, Jeffrey; Nolan, William C; Yan, Sherry X; Galvin, James E

2008-04-01

Aggregation of amyloid-beta protein (Abeta) is a key pathogenic event in Alzheimer's disease (AD). Curcumin, a constituent of the Indian spice Turmeric is structurally similar to Congo Red and has been demonstrated to bind Abeta amyloid and prevent further oligomerization of Abeta monomers onto growing amyloid beta-sheets. Reasoning that oligomerization kinetics and mechanism of amyloid formation are similar in Parkinson's disease (PD) and AD, we investigated the effect of curcumin on alpha-synuclein (AS) protein aggregation. In vitro model of AS aggregation was developed by treatment of purified AS protein (wild-type) with 1 mM Fe3+ (Fenton reaction). It was observed that the addition of curcumin inhibited aggregation in a dose-dependent manner and increased AS solubility. The aggregation-inhibiting effect of curcumin was next investigated in cell culture utilizing catecholaminergic SH-SY5Y cell line. A model system was developed in which the red fluorescent protein (DsRed2) was fused with A53T mutant of AS and its aggregation examined under different concentrations of curcumin. To estimate aggregation in an unbiased manner, a protocol was developed in which the images were captured automatically through a high-throughput cell-based screening microscope. The obtained images were processed automatically for aggregates within a defined dimension of 1-6 microm. Greater than 32% decrease in mutant alpha-synuclein aggregation was observed within 48 h subsequent to curcumin addition. Our data suggest that curcumin inhibits AS oligomerization into higher molecular weight aggregates and therefore should be further explored as a potential therapeutic compound for PD and related disorders.

Marine Synechococcus Aggregation

NASA Astrophysics Data System (ADS)

Neuer, S.; Deng, W.; Cruz, B. N.; Monks, L.

2016-02-01

Cyanobacteria are considered to play an important role in the oceanic biological carbon pump, especially in oligotrophic regions. But as single cells are too small to sink, their carbon export has to be mediated by aggregate formation and possible consumption by zooplankton producing sinking fecal pellets. Here we report results on the aggregation of the ubiquitous marine pico-cyanobacterium Synechococcus as a model organism. We first investigated the mechanism behind such aggregation by studying the potential role of transparent exopolymeric particles (TEP) and the effects of nutrient (nitrogen or phosphorus) limitation on the TEP production and aggregate formation of these pico-cyanobacteria. We further studied the aggregation and subsequent settling in roller tanks and investigated the effects of the clays kaolinite and bentonite in a series of concentrations. Our results show that despite of the lowered growth rates, Synechococcus in nutrient limited cultures had larger cell-normalized TEP production, formed a greater volume of aggregates, and resulted in higher settling velocities compared to results from replete cultures. In addition, we found that despite their small size and lack of natural ballasting minerals, Synechococcus cells could still form aggregates and sink at measureable velocities in seawater. Clay minerals increased the number and reduced the size of aggregates, and their ballasting effects increased the sinking velocity and carbon export potential of aggregates. In comparison with the Synechococcus, we will also present results of the aggregation of the pico-cyanobacterium Prochlorococcus in roller tanks. These results contribute to our understanding in the physiology of marine Synechococcus as well as their role in the ecology and biogeochemistry in oligotrophic oceans.

A 31-residue peptide induces aggregation of tau’s microtubule-binding region in cells

PubMed Central

Stöhr, Jan; Wu, Haifan; Nick, Mimi; Wu, Yibing; Bhate, Manasi; Condello, Carlo; Johnson, Noah; Rodgers, Jeffrey; Lemmin, Thomas; Achyraya, Srabasti; Becker, Julia; Robinson, Kathleen; Kelly, Mark J.S.; Gai, Feng; Stubbs, Gerald; Prusiner, Stanley B.; DeGrado, William F.

2018-01-01

The self-propagation of misfolded conformations of tau underlies neurodegenerative diseases, including Alzheimer’s disease. There is considerable interest in discovering the minimal sequence and active conformational nucleus that defines this self-propagating event. The microtubule-binding region, spanning residues 244-372, reproduces much of the aggregation behavior of tau in cells and animal models. Further dissection of the amyloid-forming region to a hexapeptide from the third microtubule-binding repeat resulted in a peptide that rapidly forms fibrils in vitro. We show here that this peptide lacks the ability to seed aggregation of tau244-372 in cells. However, as the hexapeptide is gradually extended to 31 residues, the peptides aggregate more slowly and gain potent activity to induce aggregation of tau244-372 in cells. X-ray fiber diffraction, hydrogen-deuterium exchange and solids NMR studies map the beta-forming region to a 25-residue sequence. Thus, the nucleus for self-propagating aggregation of tau244-372 in cells is packaged in a remarkably small peptide. PMID:28837163

Three cyanobacteriochromes work together to form a light color-sensitive input system for c-di-GMP signaling of cell aggregation.

PubMed

Enomoto, Gen; Ni-Ni-Win; Narikawa, Rei; Ikeuchi, Masahiko

2015-06-30

Cyanobacteriochromes (CBCRs) are cyanobacterial photoreceptors that have diverse spectral properties and domain compositions. Although large numbers of CBCR genes exist in cyanobacterial genomes, no studies have assessed whether multiple CBCRs work together. We recently showed that the diguanylate cyclase (DGC) activity of the CBCR SesA from Thermosynechococcus elongatus is activated by blue-light irradiation and that, when irradiated, SesA, via its product cyclic dimeric GMP (c-di-GMP), induces aggregation of Thermosynechococcus vulcanus cells at a temperature that is suboptimum for single-cell viability. For this report, we first characterize the photobiochemical properties of two additional CBCRs, SesB and SesC. Blue/teal light-responsive SesB has only c-di-GMP phosphodiesterase (PDE) activity, which is up-regulated by teal light and GTP. Blue/green light-responsive SesC has DGC and PDE activities. Its DGC activity is enhanced by blue light, whereas its PDE activity is enhanced by green light. A ΔsesB mutant cannot suppress cell aggregation under teal-green light. A ΔsesC mutant shows a less sensitive cell-aggregation response to ambient light. ΔsesA/ΔsesB/ΔsesC shows partial cell aggregation, which is accompanied by the loss of color dependency, implying that a nonphotoresponsive DGC(s) producing c-di-GMP can also induce the aggregation. The results suggest that SesB enhances the light color dependency of cell aggregation by degrading c-di-GMP, is particularly effective under teal light, and, therefore, seems to counteract the induction of cell aggregation by SesA. In addition, SesC seems to improve signaling specificity as an auxiliary backup to SesA/SesB activities. The coordinated action of these three CBCRs highlights why so many different CBCRs exist.

Axisymmetric drop shape analysis for estimating the surface tension of cell aggregates by centrifugation.

PubMed

Kalantarian, Ali; Ninomiya, Hiromasa; Saad, Sameh M I; David, Robert; Winklbauer, Rudolf; Neumann, A Wilhelm

2009-02-18

Biological tissues behave in certain respects like liquids. Consequently, the surface tension concept can be used to explain aspects of the in vitro and in vivo behavior of multicellular aggregates. Unfortunately, conventional methods of surface tension measurement cannot be readily applied to small cell aggregates. This difficulty can be overcome by an experimentally straightforward method consisting of centrifugation followed by axisymmetric drop shape analysis (ADSA). Since the aggregates typically show roughness, standard ADSA cannot be applied and we introduce a novel numerical method called ADSA-IP (ADSA for imperfect profile) for this purpose. To examine the new methodology, embryonic tissues from the gastrula of the frog, Xenopus laevis, deformed in the centrifuge are used. It is confirmed that surface tension measurements are independent of centrifugal force and aggregate size. Surface tension is measured for ectodermal cells in four sample batches, and varies between 1.1 and 7.7 mJ/m2. Surface tension is also measured for aggregates of cells expressing cytoplasmically truncated EP/C-cadherin, and is approximately half as large. In parallel, such aggregates show a reduction in convergent extension-driven elongation after activin treatment, reflecting diminished intercellular cohesion.

Alpha-synuclein aggregates activate calcium pump SERCA leading to calcium dysregulation.

PubMed

Betzer, Cristine; Lassen, Louise Berkhoudt; Olsen, Anders; Kofoed, Rikke Hahn; Reimer, Lasse; Gregersen, Emil; Zheng, Jin; Calì, Tito; Gai, Wei-Ping; Chen, Tong; Moeller, Arne; Brini, Marisa; Fu, Yuhong; Halliday, Glenda; Brudek, Tomasz; Aznar, Susana; Pakkenberg, Bente; Andersen, Jens Peter; Jensen, Poul Henning

2018-05-01

Aggregation of α-synuclein is a hallmark of Parkinson's disease and dementia with Lewy bodies. We here investigate the relationship between cytosolic Ca 2+ and α-synuclein aggregation. Analyses of cell lines and primary culture models of α-synuclein cytopathology reveal an early phase with reduced cytosolic Ca 2+ levels followed by a later Ca 2+ increase. Aggregated but not monomeric α-synuclein binds to and activates SERCA in vitro , and proximity ligation assays confirm this interaction in cells. The SERCA inhibitor cyclopiazonic acid (CPA) normalises both the initial reduction and the later increase in cytosolic Ca 2+ CPA protects the cells against α-synuclein-aggregate stress and improves viability in cell models and in Caenorhabditis elegans in vivo Proximity ligation assays also reveal an increased interaction between α-synuclein aggregates and SERCA in human brains affected by dementia with Lewy bodies. We conclude that α-synuclein aggregates bind SERCA and stimulate its activity. Reducing SERCA activity is neuroprotective, indicating that SERCA and down-stream processes may be therapeutic targets for treating α-synucleinopathies. © 2018 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

Detection and characterization of red blood cell (RBC) aggregation with photoacoustics

NASA Astrophysics Data System (ADS)

Hysi, Eno; Saha, Ratan K.; Rui, Min; Kolios, Michael C.

2012-02-01

Red blood cells (RBCs) aggregate in the presence of increased plasma fibrinogen and low shear forces during blood flow. RBC aggregation has been observed in deep vein thrombosis, sepsis and diabetes. We propose using photoacoustics (PA) as a non-invasive imaging modality to detect RBC aggregation. The theoretical and experimental feasibility of PA for detecting and characterizing aggregation was assessed. A simulation study was performed to generate PA signals from non-aggregated and aggregated RBCs using a frequency domain approach and to study the PA signals' dependence on hematocrit and aggregate size. The effect of the finite bandwidth nature of transducers on the PA power spectra was also investigated. Experimental confirmation of theoretical results was conducted using porcine RBC samples exposed to 1064 nm optical wavelength using the Imagio Small Animal PA imaging system (Seno Medical Instruments, Inc., San Antonio, TX). Aggregation was induced with Dextran-70 (Sigma-Aldrich, St. Louis, MO) and the effect of hematocrit and aggregation level was investigated. The theoretical and experimental PA signal amplitude increased linearly with increasing hematocrit. The theoretical dominant frequency content of PA signals shifted towards lower frequencies (<30 MHz) and 9 dB enhancements in spectral power were observed as the size of aggregates increased compared to non-aggregating RBCs. Calibration of the PA spectra with the transducer response obtained from a 200 nm gold film was performed to remove system dependencies. Analysis of the spectral parameters from the calibrated spectra suggested that PA can assess the degree of aggregation at multiple hematocrit and aggregation levels.

Coherent and incoherent ultrasound backscatter from cell aggregates.

PubMed

de Monchy, Romain; Destrempes, François; Saha, Ratan K; Cloutier, Guy; Franceschini, Emilie

2016-09-01

The effective medium theory (EMT) was recently developed to model the ultrasound backscatter from aggregating red blood cells [Franceschini, Metzger, and Cloutier, IEEE Trans. Ultrason. Ferroelectr. Freq. Control 58, 2668-2679 (2011)]. The EMT assumes that aggregates can be treated as homogeneous effective scatterers, which have effective properties determined by the aggregate compactness and the acoustical characteristics of the cells and the surrounding medium. In this study, the EMT is further developed to decompose the differential backscattering cross section of a single cell aggregate into coherent and incoherent components. The coherent component corresponds to the squared norm of the average scattering amplitude from the effective scatterer, and the incoherent component considers the variance of the scattering amplitude (i.e., the mean squared norm of the fluctuation of the scattering amplitude around its mean) within the effective scatterer. A theoretical expression for the incoherent component based on the structure factor is proposed and compared with another formulation based on the Gaussian direct correlation function. This theoretical improvement is assessed using computer simulations of ultrasound backscatter from aggregating cells. The consideration of the incoherent component based on the structure factor allows us to approximate the simulations satisfactorily for a product of the wavenumber times the aggregate radius kr ag around 2.

Transient inter-cellular polymeric linker.

PubMed

Ong, Siew-Min; He, Lijuan; Thuy Linh, Nguyen Thi; Tee, Yee-Han; Arooz, Talha; Tang, Guping; Tan, Choon-Hong; Yu, Hanry

2007-09-01

Three-dimensional (3D) tissue-engineered constructs with bio-mimicry cell-cell and cell-matrix interactions are useful in regenerative medicine. In cell-dense and matrix-poor tissues of the internal organs, cells support one another via cell-cell interactions, supplemented by small amount of the extra-cellular matrices (ECM) secreted by the cells. Here we connect HepG2 cells directly but transiently with inter-cellular polymeric linker to facilitate cell-cell interaction and aggregation. The linker consists of a non-toxic low molecular-weight polyethyleneimine (PEI) backbone conjugated with multiple hydrazide groups that can aggregate cells within 30 min by reacting with the aldehyde handles on the chemically modified cell-surface glycoproteins. The cells in the cellular aggregates proliferated; and maintained the cortical actin distribution of the 3D cell morphology while non-aggregated cells died over 7 days of suspension culture. The aggregates lost distinguishable cell-cell boundaries within 3 days; and the ECM fibers became visible around cells from day 3 onwards while the inter-cellular polymeric linker disappeared from the cell surfaces over time. The transient inter-cellular polymeric linker can be useful for forming 3D cellular and tissue constructs without bulk biomaterials or extensive network of engineered ECM for various applications.

Role of Carbonyl Modifications on Aging-Associated Protein Aggregation

PubMed Central

Tanase, Maya; Urbanska, Aleksandra M.; Zolla, Valerio; Clement, Cristina C.; Huang, Liling; Morozova, Kateryna; Follo, Carlo; Goldberg, Michael; Roda, Barbara; Reschiglian, Pierluigi; Santambrogio, Laura

2016-01-01

Protein aggregation is a common biological phenomenon, observed in different physiological and pathological conditions. Decreased protein solubility and a tendency to aggregate is also observed during physiological aging but the causes are currently unknown. Herein we performed a biophysical separation of aging-related high molecular weight aggregates, isolated from the bone marrow and splenic cells of aging mice and followed by biochemical and mass spectrometric analysis. The analysis indicated that compared to younger mice an increase in protein post-translational carbonylation was observed. The causative role of these modifications in inducing protein misfolding and aggregation was determined by inducing carbonyl stress in young mice, which recapitulated the increased protein aggregation observed in old mice. Altogether our analysis indicates that oxidative stress-related post-translational modifications accumulate in the aging proteome and are responsible for increased protein aggregation and altered cell proteostasis. PMID:26776680

Hydrodynamic size-based separation and characterization of protein aggregates from total cell lysates

PubMed Central

Tanase, Maya; Zolla, Valerio; Clement, Cristina C; Borghi, Francesco; Urbanska, Aleksandra M; Rodriguez-Navarro, Jose Antonio; Roda, Barbara; Zattoni, Andrea; Reschiglian, Pierluigi; Cuervo, Ana Maria; Santambrogio, Laura

2016-01-01

Herein we describe a protocol that uses hollow-fiber flow field-flow fractionation (FFF) coupled with multiangle light scattering (MALS) for hydrodynamic size-based separation and characterization of complex protein aggregates. The fractionation method, which requires 1.5 h to run, was successfully modified from the analysis of protein aggregates, as found in simple protein mixtures, to complex aggregates, as found in total cell lysates. In contrast to other related methods (filter assay, analytical ultracentrifugation, gel electrophoresis and size-exclusion chromatography), hollow-fiber flow FFF coupled with MALS allows a flow-based fractionation of highly purified protein aggregates and simultaneous measurement of their molecular weight, r.m.s. radius and molecular conformation (e.g., round, rod-shaped, compact or relaxed). The polyethersulfone hollow fibers used, which have a 0.8-mm inner diameter, allow separation of as little as 20 μg of total cell lysates. In addition, the ability to run the samples in different denaturing and nondenaturing buffer allows defining true aggregates from artifacts, which can form during sample preparation. The protocol was set up using Paraquat-induced carbonylation, a model that induces protein aggregation in cultured cells. This technique will advance the biochemical, proteomic and biophysical characterization of molecular-weight aggregates associated with protein mutations, as found in many CNS degenerative diseases, or chronic oxidative stress, as found in aging, and chronic metabolic and inflammatory conditions. PMID:25521790

Dynamics of mutated GFAP aggregates revealed by real-time imaging of an astrocyte model of Alexander disease.

PubMed

Mignot, Cyril; Delarasse, Cécile; Escaich, Séverine; Della Gaspera, Bruno; Noé, Eric; Colucci-Guyon, Emma; Babinet, Charles; Pekny, Milos; Vicart, Patrick; Boespflug-Tanguy, Odile; Dautigny, André; Rodriguez, Diana; Pham-Dinh, Danielle

2007-08-01

Alexander disease (AxD) is a rare neurodegenerative disorder characterized by large cytoplasmic aggregates in astrocytes and myelin abnormalities and caused by dominant mutations in the gene encoding glial fibrillary acidic protein (GFAP), the main intermediate filament protein in astrocytes. We tested the effects of three mutations (R236H, R76H and L232P) associated with AxD in cells transiently expressing mutated GFAP fused to green fluorescent protein (GFP). Mutated GFAP-GFP expressed in astrocytes formed networks or aggregates similar to those found in the brains of patients with the disease. Time-lapse recordings of living astrocytes showed that aggregates of mutated GFAP-GFP may either disappear, associated with cell survival, or coalesce in a huge juxtanuclear structure associated with cell death. Immunolabeling of fixed cells suggested that this gathering of aggregates forms an aggresome-like structure. Proteasome inhibition and immunoprecipitation assays revealed mutated GFAP-GFP ubiquitination, suggesting a role of the ubiquitin-proteasome system in the disaggregation process. In astrocytes from wild-type-, GFAP-, and vimentin-deficient mice, mutated GFAP-GFP aggregated or formed a network, depending on qualitative and quantitative interactions with normal intermediate filament partners. Particularly, vimentin displayed an anti-aggregation effect on mutated GFAP. Our data indicate a dynamic and reversible aggregation of mutated GFAP, suggesting that therapeutic approaches may be possible.

Cellular Strategies for Regulating Functional and Nonfunctional Protein Aggregation

PubMed Central

Gsponer, Jörg; Babu, M. Madan

2012-01-01

Summary Growing evidence suggests that aggregation-prone proteins are both harmful and functional for a cell. How do cellular systems balance the detrimental and beneficial effect of protein aggregation? We reveal that aggregation-prone proteins are subject to differential transcriptional, translational, and degradation control compared to nonaggregation-prone proteins, which leads to their decreased synthesis, low abundance, and high turnover. Genetic modulators that enhance the aggregation phenotype are enriched in genes that influence expression homeostasis. Moreover, genes encoding aggregation-prone proteins are more likely to be harmful when overexpressed. The trends are evolutionarily conserved and suggest a strategy whereby cellular mechanisms specifically modulate the availability of aggregation-prone proteins to (1) keep concentrations below the critical ones required for aggregation and (2) shift the equilibrium between the monomeric and oligomeric/aggregate form, as explained by Le Chatelier’s principle. This strategy may prevent formation of undesirable aggregates and keep functional assemblies/aggregates under control. PMID:23168257

Hypoxic Three-Dimensional Scaffold-Free Aggregate Cultivation of Mesenchymal Stem Cells in a Stirred Tank Reactor.

PubMed

Egger, Dominik; Schwedhelm, Ivo; Hansmann, Jan; Kasper, Cornelia

2017-05-23

Extensive expansion of mesenchymal stem cells (MSCs) for cell-based therapies remains challenging since long-term cultivation and excessive passaging in two-dimensional conditions result in a loss of essential stem cell properties. Indeed, low survival rate of cells, alteration of surface marker profiles, and reduced differentiation capacity are observed after in vitro expansion and reduce therapeutic success in clinical studies. Remarkably, cultivation of MSCs in three-dimensional aggregates preserve stem cell properties. Hence, the large scale formation and cultivation of MSC aggregates is highly desirable. Besides other effects, MSCs cultivated under hypoxic conditions are known to display increased proliferation and genetic stability. Therefore, in this study we demonstrate cultivation of adipose derived human MSC aggregates in a stirred tank reactor under hypoxic conditions. Although aggregates were exposed to comparatively high average shear stress of 0.2 Pa as estimated by computational fluid dynamics, MSCs displayed a viability of 78-86% and maintained their surface marker profile and differentiation potential after cultivation. We postulate that cultivation of 3D MSC aggregates in stirred tank reactors is valuable for large-scale production of MSCs or their secreted compounds after further optimization of cultivation parameters.

Antimicrobial and anticancer activity of AgNPs coated with Alphonsea sclerocarpa extract.

PubMed

Doddapaneni, Suman Joshi D S; Amgoth, Chander; Kalle, Arunasree M; Suryadevara, Surya Narayana; Alapati, Krishna Satya

2018-03-01

The synthesis and characterization of an aggregate of AgNPs coated with plant extract (PE) from Alphonsea sclerocarpa and its significant antimicrobial activity and inhibition on K562 (blood cancer) cells have been appended in the article. Synthesis of aggregate [(AgNPs)-(PE)] has been followed by a facile eco-friendly approach without using any harmful chemicals. The morphology of an aggregate [(AgNPs)-(PE)] was confirmed by TEM and SEM microscopic characterizations. Properties like solid state, the presence of functional groups, and elemental composition have been characterized through the XRD, FTIR, and EDAX. The biocompatibility of synthesized aggregate of [(AgNPs)-(PE)] was confirmed by the MTT assay. An in vitro cell (HEK293)-based studies were performed for the biocompatibility tests and it is found that the aggregate [(AgNPs)-(PE)] is not harmful to normal/healthy cells. Even though A. sclerocarpa show the antimicrobial (antibacterial and antifungal) activity, it has been further enhanced with the developed aggregate of [(AgNPs)-(PE)]. Furthermore, it has been extended to examine the cellular inhibition on K562 cells and obtained > 75% cell inhibition for 24 h treated cells.

A phase field approach for multicellular aggregate fusion in biofabrication.

PubMed

Yang, Xiaofeng; Sun, Yi; Wang, Qi

2013-07-01

We present a modeling and computational approach to study fusion of multicellular aggregates during tissue and organ fabrication, which forms the foundation for the scaffold-less biofabrication of tissues and organs known as bioprinting. It is known as the phase field method, where multicellular aggregates are modeled as mixtures of multiphase complex fluids whose phase mixing or separation is governed by interphase force interactions, mimicking the cell-cell interaction in the multicellular aggregates, and intermediate range interaction mediated by the surrounding hydrogel. The material transport in the mixture is dictated by hydrodynamics as well as forces due to the interphase interactions. In a multicellular aggregate system with fixed number of cells and fixed amount of the hydrogel medium, the effect of cell differentiation, proliferation, and death are neglected in the current model, which can be readily included in the model, and the interaction between different components is dictated by the interaction energy between cell and cell as well as between cell and medium particles, respectively. The modeling approach is applicable to transient simulations of fusion of cellular aggregate systems at the time and length scale appropriate to biofabrication. Numerical experiments are presented to demonstrate fusion and cell sorting during tissue and organ maturation processes in biofabrication.

Radiation damage to the microvasculature in the rabbit ear chamber. An electron microscope study. [X radiation

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

Yang, V.V.; Stearner, S.P.; Dimitrievich, G.S.

1977-04-01

Cell aggregates in increased numbers appear along blood vessel walls within a few days after local x irradiation of the tissue within rabbit ear chambers. At 7 days after irradiation with 400 or 700 rad of 250 kVp of x rays, electron microscopic studies of the microvasculature were carried out to determine the morphological characteristics of the cell types involved in the aggregates and the relation of these cells to vascular repair. The cell aggregates usually occur in the interstitial region subjacent to the endothelium. The cells that make up the aggregates show morphological characteristics of relatively undifferentiated mesenchymal cells;more » they have an irregularly rounded shape and contain large amounts of rough endoplasmic reticulum, Golgi vesicles, and mitochondria. In a few instances, cells of similar morphology also occur as part of the lining of the blood vessels. The perivascular cell aggregates may originate from the pericyte population or from undifferentiated mesenchymal cells that occur in the interstitial region surrounding blood vessels; it is improbable that they are dedifferentiated smooth muscle cells. It is suggested that the cells that make up these aggregates contribute to the repair of the microvasculation after radiation injury. The radiosensitivity of vascular endothelium reported by previous investigators seems to preclude endothelial proliferation as the principal repair mechanism at higher radiation doses.« less

The small heat shock proteins αB-crystallin (HSPB5) and Hsp27 (HSPB1) inhibit the intracellular aggregation of α-synuclein.

PubMed

Cox, Dezerae; Ecroyd, Heath

2017-07-01

Protein homeostasis, or proteostasis, is the process of maintaining the conformational and functional integrity of the proteome. Proteostasis is preserved in the face of stress by a complex network of cellular machinery, including the small heat shock molecular chaperone proteins (sHsps), which act to inhibit the aggregation and deposition of misfolded protein intermediates. Despite this, the pathogenesis of several neurodegenerative diseases has been inextricably linked with the amyloid fibrillar aggregation and deposition of α-synuclein (α-syn). The sHsps are potent inhibitors of α-syn aggregation in vitro. However, the limited availability of a robust, cell-based model of α-syn aggregation has, thus far, restricted evaluation of sHsp efficacy in the cellular context. As such, this work sought to establish a robust model of intracellular α-syn aggregation using Neuro-2a cells. Aggregation of α-syn was found to be sensitive to inhibition of autophagy and the proteasome, resulting in a significant increase in the proportion of cells containing α-syn inclusions. This model was then used to evaluate the capacity of the sHsps, αB-c and Hsp27, to prevent α-syn aggregation in cells. To do so, we used bicistronic expression plasmids to express the sHsps. Unlike traditional fluorescent fusion constructs, these bicistronic expression plasmids enable only individual transfected cells expressing the sHsps (via expression of the fluorescent reporter) to be analysed, but without the need to tag the sHsp, which can affect its oligomeric structure and chaperone activity. Overexpression of both αB-c and Hsp27 significantly reduced the intracellular aggregation of α-syn. Thus, these findings suggest that overexpressing or boosting the activity of sHsps may be a way of preventing amyloid fibrillar aggregation of α-syn in the context of neurodegenerative disease.

Elucidation of flow-mediated tumour cell-induced platelet aggregation using an ultrasound standing wave trap.

PubMed

Bazou, D; Santos-Martinez, M J; Medina, C; Radomski, M W

2011-04-01

Tumour cells activate and aggregate platelets [tumour cell-induced platelet aggregation (TCIPA)] and this process plays an important role in the successful metastasis of cancer cells. To date, most studies on TCIPA have been conducted under no-flow conditions. In this study, we have investigated TCIPA in real time under flow conditions, using an ultrasound standing wave trap that allows formation and levitation of cancer cell clusters in suspension, thus mimicking the conditions generated by flowing blood. Using 59M adenocarcinoma and HT1080 fibrosarcoma cells and human platelets, cancer cell cluster-platelet aggregates were imaged in real time using epi-fluorescence microscopy (F-actin) and investigated in detail using confocal microscopy (matrix metalloproteinase-2-GPIIb/IIIa co-localization) and scanning electron and helium-ion microscopy (<1 nm resolution). The release of gelatinases from aggregates was studied using zymography. We found that platelet activation and aggregation takes place on the surface of cancer cells (TCIPA), leading to time-dependent disruption of cancer cell clusters. Pharmacological modulation of TCIPA revealed that EDTA, prostacyclin, o-phenanthroline and apyrase significantly down-regulated TCIPA and, in turn, delayed cell cluster disruption, However, EGTA and aspirin were ineffective. Pharmacological inhibition of TCIPA correlated with the down-regulation of platelet activation as shown by flow-cytometry assay of platelet P-selectin. Our results show for the first time, that during TCIPA, platelet activation disrupts cancer cell clusters and this can contribute to metastasis. Thus, selective targeting of platelet aggregate-cancer cell clusters may be an important strategy to control metastasis. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

Genipin-crosslinked microcarriers mediating hepatocellular aggregates formation and functionalities.

PubMed

Lau, Ting Ting; Wang, Chunming; Png, Sze Wei; Su, Kai; Wang, Dong-An

2011-01-01

In engineered regenerative medicine, various types of scaffolds have been customized to pursue the optimal environment for different types of therapeutic cells. In liver therapeutic research, hepatocytes require attachment to solid anchors for survival and proliferation before they could grow into cellular aggregates with enhanced functionalities. Among the various biomaterials scaffolds and vehicles, microspherical cell carriers are suited to these requirements. Individual spheres may provide two-dimensional (2D) cell-affinitive surfaces for cell adhesion and spreading; whereas multiple microcarriers may form three-dimensional (3D) matrices with inter-spherical space for cell expansion and multicellular aggregation. In this study, we culture human liver carcinoma cell line (HepG2) cells on genipin-crosslinked gelatin microspheres of two different sizes. Results suggest that both microcarriers support cell adhesion, proliferation, and spontaneous formation of hepatocellular aggregates, among which the spheres with bigger size (200-300 μm) seem more favorable than the smaller ones in terms of aggregate formation and liver specific functionalities. These findings suggest that the genipin-crosslinked microcarrier is a competent vehicle for liver cell delivery. Copyright © 2010 Wiley Periodicals, Inc.

Involvement of cell surface TG2 in the aggregation of K562 cells triggered by gluten.

PubMed

Feriotto, G; Calza, R; Bergamini, C M; Griffin, M; Wang, Z; Beninati, S; Ferretti, V; Marzola, E; Guerrini, R; Pagnoni, A; Cavazzini, A; Casciano, F; Mischiati, C

2017-03-01

Gluten-induced aggregation of K562 cells represents an in vitro model reproducing the early steps occurring in the small bowel of celiac patients exposed to gliadin. Despite the clear involvement of TG2 in the activation of the antigen-presenting cells, it is not yet clear in which compartment it occurs. Herein we study the calcium-dependent aggregation of these cells, using either cell-permeable or cell-impermeable TG2 inhibitors. Gluten induces efficient aggregation when calcium is absent in the extracellular environment, while TG2 inhibitors do not restore the full aggregating potential of gluten in the presence of calcium. These findings suggest that TG2 activity is not essential in the cellular aggregation mechanism. We demonstrate that gluten contacts the cells and provokes their aggregation through a mechanism involving the A-gliadin peptide 31-43. This peptide also activates the cell surface associated extracellular TG2 in the absence of calcium. Using a bioinformatics approach, we identify the possible docking sites of this peptide on the open and closed TG2 structures. Peptide docks with the closed TG2 structure near to the GTP/GDP site, by establishing molecular interactions with the same amino acids involved in stabilization of GTP binding. We suggest that it may occur through the displacement of GTP, switching the TG2 structure from the closed to the active open conformation. Furthermore, docking analysis shows peptide binding with the β-sandwich domain of the closed TG2 structure, suggesting that this region could be responsible for the different aggregating effects of gluten shown in the presence or absence of calcium. We deduce from these data a possible mechanism of action by which gluten makes contact with the cell surface, which could have possible implications in the celiac disease onset.

Myxobacteria Fruiting Body Formation

NASA Astrophysics Data System (ADS)

Jiang, Yi

2006-03-01

Myxobacteria are social bacteria that swarm and glide on surfaces, and feed cooperatively. When starved, tens of thousands of cells change their movement pattern from outward spreading to inward concentration; they form aggregates that become fruiting bodies, inside which cells differentiate into nonmotile, environmentally resistant spores. Traditionally, cell aggregation has been considered to imply chemotaxis, a long-range cell interaction mediated by diffusing chemicals. However, myxobacteria aggregation is the consequence of direct cell-contact interactions. I will review our recent efforts in modeling the fruiting body formation of Myxobacteria, using lattice gas cellular automata models that are based on local cell-cell contact signaling. These models have reproduced the individual phases in Myxobacteria development such as the rippling, streaming, early aggregation and the final sporulation; the models can be unified to simulate the whole developmental process of Myxobacteria.

Occurrence of lymphohaemopoietic tissue in the meninges of the stingray Dasyatis akajei (Elasmobranchii, chondricthyes).

PubMed

Chiba, A; Torroba, M; Honma, Y; Zapata, A G

1988-11-01

The cytoarchitecture of the lymphohaemopoietic masses occurring in the "meninx primitiva" of the stingray Dasyatis akajei (Elasmobranchii, Chondricthyes) has been analyzed by light and scanning and transmission electron microscopy. Lymphohaemopoietic aggregates showing similar morphologies occurred along all the central nervous system, but they were more frequent in the telencephalon, diencephalon, and mesencephalon. In each aggregate, the granulopoietic tissue appeared in a fibroblastic stroma surrounding the large blood vessels, and the lymphoid components were present in a reticular network. Developing and mature eosinophils and heterophils--as well as lymphocytes, monocytes, macrophages, and plasma cells--are the main free cells present in these meningeal aggregates. The remarkable intimate association between macrophages and lymphoid cells to form close cell clusters suggests some immunological capacity for the meningeal lymphohaemopoietic tissue. According to their capacities, presence of lymphoid tissue, and histological organization, the meningeal lymphohemopoietic aggregates of Dasyatis akajei resemble other lymphomyeloid aggregates associated with cranium and choroid plexuses in Holocephali and Ganoidei. The phylogenetical relationships of these aggregates with mammalian bone marrow are discussed.

Label-free detection of aggregated platelets in blood by machine-learning-aided optofluidic time-stretch microscopy.

PubMed

Jiang, Yiyue; Lei, Cheng; Yasumoto, Atsushi; Kobayashi, Hirofumi; Aisaka, Yuri; Ito, Takuro; Guo, Baoshan; Nitta, Nao; Kutsuna, Natsumaro; Ozeki, Yasuyuki; Nakagawa, Atsuhiro; Yatomi, Yutaka; Goda, Keisuke

2017-07-11

According to WHO, about 10 million new cases of thrombotic disorders are diagnosed worldwide every year. Thrombotic disorders, including atherothrombosis (the leading cause of death in the US and Europe), are induced by occlusion of blood vessels, due to the formation of blood clots in which aggregated platelets play an important role. The presence of aggregated platelets in blood may be related to atherothrombosis (especially acute myocardial infarction) and is, hence, useful as a potential biomarker for the disease. However, conventional high-throughput blood analysers fail to accurately identify aggregated platelets in blood. Here we present an in vitro on-chip assay for label-free, single-cell image-based detection of aggregated platelets in human blood. This assay builds on a combination of optofluidic time-stretch microscopy on a microfluidic chip operating at a high throughput of 10 000 blood cells per second with machine learning, enabling morphology-based identification and enumeration of aggregated platelets in a short period of time. By performing cell classification with machine learning, we differentiate aggregated platelets from single platelets and white blood cells with a high specificity and sensitivity of 96.6% for both. Our results indicate that the assay is potentially promising as predictive diagnosis and therapeutic monitoring of thrombotic disorders in clinical settings.

Auto-aggregation properties of a novel aerobic denitrifier Enterobacter sp. strain FL.

PubMed

Wang, Xia; An, Qiang; Zhao, Bin; Guo, Jin Song; Huang, Yuan Sheng; Tian, Meng

2018-02-01

Enterobacter sp. strain FL was newly isolated from activated sludge and exhibited significant capability of auto-aggregation as well as aerobic denitrification. The removal efficiencies of NO 3 - -N, total nitrogen (TN), and TOC by strain FL in batch culture reached 94.6, 63.9, and 72.5% in 24 h, respectively. The production of N 2 O and N 2 in the presence of oxygen demonstrated the occurrence of aerobic denitrification. The auto-aggregation index of strain FL reached 54.3%, suggesting a high tendency that the cells would agglomerate into aggregates. The production of extracellular polymeric substances (EPSs), which were mainly composed of proteins followed by polysaccharides, was considered to be related to the cell aggregation according to Fourier transform infrared (FT-IR) and confocal laser scanning microscopy (CLSM). The proteins in EPS were evenly and tightly combined to cells and altered the protein secondary structures of cell surface from random coils to β-sheets and three-turn helices. The alteration of protein secondary structures of cell surface caused by the proteins in EPS might play a dominant role in the auto-aggregation of strain FL. To further assess the feasibility of strain FL for synthetic wastewater treatment, a sequencing batch reactor (SBR), solely inoculated with strain FL, was conducted. During the 16 running cycles, the removal efficiency of NO 3 - -N was 90.2-99.7% and the auto-aggregation index was stabilized at 35.0-41.5%. The EPS promoted the biomass of strain FL to aggregate in the SBR.

Effect of salivary agglutination on oral streptococcal clearance by human polymorphonuclear neutrophil granulocytes

PubMed Central

Itzek, Andreas; Chen, Zhiyun; Merritt, Justin; Kreth, Jens

2016-01-01

Salivary agglutination is an important host defense mechanism to aggregate oral commensal bacteria as well as invading pathogens. Saliva flow and subsequent swallowing more easily clear aggregated bacteria compared to single cells. Phagocytic clearance of bacteria through polymorphonuclear neutrophil granulocytes also seems to increase to a certain extent with the size of bacterial aggregates. To determine a connection between salivary agglutination and the host innate immune response by phagocytosis, an in vitro agglutination assay was developed reproducing the average size of salivary bacterial aggregates. Using the oral commensal Streptococcus gordonii as a model organism, the effect of salivary agglutination to the phagocytic clearance through polymorphonuclear neutrophil granulocytes was investigated. Here we describe that salivary aggregates of S. gordonii are readily cleared through phagocytosis, while single bacterial cells showed a significant delay in being phagocytosed and killed. Furthermore, prior to phagocytosis the polymorphonuclear neutrophil granulocytes were able to induce a specific de-aggregation, which was dependent on serine protease activity. The herein presented data suggest that salivary agglutination of bacterial cells leads to an ideal size for recognition by polymorphonuclear neutrophil granulocytes. As a first line of defense, these phagocytic cells are able to recognize the aggregates and de-aggregate them via serine proteases to a more manageable size for efficient phagocytosis and subsequent killing in the phagolysosome. This observed mechanism not only prevents the rapid spreading of oral bacterial cells while entering the bloodstream but would also avoid degranulation of involved polymorphonuclear neutrophil granulocytes thus preventing collateral damage to nearby tissue. PMID:27194631

Effect of salivary agglutination on oral streptococcal clearance by human polymorphonuclear neutrophil granulocytes.

PubMed

Itzek, A; Chen, Z; Merritt, J; Kreth, J

2017-06-01

Salivary agglutination is an important host defense mechanism to aggregate oral commensal bacteria as well as invading pathogens. Saliva flow and subsequent swallowing more easily clear aggregated bacteria compared with single cells. Phagocytic clearance of bacteria through polymorphonuclear neutrophil granulocytes also seems to increase to a certain extent with the size of bacterial aggregates. To determine a connection between salivary agglutination and the host innate immune response by phagocytosis, an in vitro agglutination assay was developed reproducing the average size of salivary bacterial aggregates. Using the oral commensal Streptococcus gordonii as a model organism, the effect of salivary agglutination on phagocytic clearance through polymorphonuclear neutrophil granulocytes was investigated. Here we describe how salivary aggregates of S. gordonii are readily cleared through phagocytosis, whereas single bacterial cells showed a significant delay in being phagocytosed and killed. Furthermore, before phagocytosis the polymorphonuclear neutrophil granulocytes were able to induce a specific de-aggregation, which was dependent on serine protease activity. The data presented suggest that salivary agglutination of bacterial cells leads to an ideal size for recognition by polymorphonuclear neutrophil granulocytes. As a first line of defense, these phagocytic cells are able to recognize the aggregates and de-aggregate them via serine proteases to a more manageable size for efficient phagocytosis and subsequent killing in the phagolysosome. This observed mechanism not only prevents the rapid spreading of oral bacterial cells while entering the bloodstream but would also avoid degranulation of involved polymorphonuclear neutrophil granulocytes, so preventing collateral damage to nearby tissue. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Aggregation of Human Eyelid Adipose-derived Stem Cells by Human Body Fluids

PubMed Central

Song, Yeonhwa; Yun, Sujin; Yang, Hye Jin; Yoon, A Young; Kim, Haekwon

2012-01-01

Fetal bovine serum (FBS) is the most frequently used serum for the cultivation of mammalian cells. However, since animal-derived materials might not be appropriate due to safety issues, allogeneic human serum (HS) has been used to replace FBS, particularly for the culture of human cells. While there has been a debate about the advantages of HS, its precise effect on human adult stem cells have not been clarified. The present study aimed to investigate the effect of HS on the human eyelid adipose stem cells (HEACs) in vitro. When HEACs were cultivated in a medium containing 10% HS, many cells moved into several spots and aggregated there. The phenomenon was observed as early as 9 days following 10% HS treatment, and 12 days following 5% HS plus 5% FBS treatment. However, the aggregation was never observed when the same cells were cultivated with 10% FBS or bovine serum albumin. To examine whether cell density might affect the aggregation, cells were seeded with different densities on 12-well dish. Until the beginning of aggregation, cells seeded at low densities exhibited the longest culture period of 16 days whereas cells seeded at high densities showed the shortest period of 9 days to form aggregation. The number of cells was 15.1±0.2×104 as the least for the low density group, and 29.3±2.8×104 as the greatest for the high density group. When human cord blood serum or normal bovine serum was examined for the same effect on HEACs, interestingly, cord blood serum induced the aggregation of cells whereas bovine serum treatment has never induced. When cells were cultivated with 10% HS for 9 days, they were obtained and analyzed by RT-PCR. Compared to FBS-cultivated HEACs, HS-cultivated HEACs did not express VIM, and less expressed GATA4, PALLD. On the other hand, HS-cultivated HEACs expressed MAP2 more than FBS-cultivated HEACs. In conclusion, human adult stem cells could move and form aggregates by the treatment with human body fluids. PMID:25949109

Aggregation of Culture Expanded Human Mesenchymal Stem Cells in Microcarrier-based Bioreactor.

PubMed

Yuan, Xuegang; Tsai, Ang-Chen; Farrance, Iain; Rowley, Jon; Ma, Teng

2018-03-15

Three-dimensional aggregation of human mesenchymal stem cells (hMSCs) has been used to enhance their therapeutic properties but current fabrication protocols depend on laboratory methods and are not scalable. In this study, we developed thermal responsive poly(N-isopropylacrylamide) grafted microcarriers (PNIPAM-MCs), which supported expansion and thermal detachment of hMSCs at reduced temperature (23.0 °C). hMSCs were cultured on the PNIPAM-MCs in both spinner flask (SF) and PBS Vertical-Wheel (PBS-VW) bioreactors for expansion. At room temperature, hMSCs were detached as small cell sheets, which subsequently self-assembled into 3D hMSC aggregates in PBS-VW bioreactor and remain as single cells in SF bioreactor owing to different hydrodynamic conditions. hMSC aggregates generated from the bioreactor maintained comparable immunomodulation and cytokine secretion properties compared to the ones made from the AggreWell ® . The results of the current study demonstrate the feasibility of scale-up production of hMSC aggregates in the suspension bioreactor using thermal responsive microcarriers for integrated cell expansion and 3D aggregation in a close bioreactor system and highlight the critical role of hydrodynamics in self-assembly of detached hMSC in suspension.

Polyvinylpyrrolidone- (PVP-) coated silver aggregates for high performance surface-enhanced Raman scattering in living cells.

PubMed

Tan, Xuebin; Wang, Zhuyuan; Yang, Jing; Song, Chunyuan; Zhang, Ruohu; Cui, Yiping

2009-11-04

A biocompatible and stable surface-enhanced Raman scattering (SERS) probe has been successfully synthesized through a simple route with silver aggregates. Polyvinylpyrrolidone (PVP), a biocompatible polymer, was utilized to control the aggregation process and improve the chemical stability of the aggregates. Extinction spectroscopy and TEM results show the aggregation degree and core-shell structure of the probe. It is found that when we employ 4-mercaptobenzoic acid (4MBA), crystal violet (CV), Rhodamine 6G (R6G) or 4,4'-bipyridine molecules as Raman reporters, the SERS signal from the proposed probe can remain at a high level under aggressive chemical environments, even after being incorporated into living cells. In comparison with the traditional probes without the PVP shell, the new ones exhibit strong surface-enhanced effects and low toxicity towards living cells. We demonstrate that the PVP-coated silver aggregates are highly SERS effective, for which the fabrication protocol is advantageous in its simplicity and reproducibility.

[A study of the aggregation of human red blood cells induced by picric acid].

PubMed

Sheremet'ev, Iu A; Sheremet'eva, A V; Lednev, A V

2005-01-01

The effect of picric acid on the aggregation of human erythrocytes was studied. It was shown that the addition of picric acid to a suspension of washed erythrocytes leads to a decrease in pH of medium to 1.5-2 and the formation of echinocytes. Stirring the suspension of echinocytes at low pH values results in a strong aggregation of cells. Increasing the pH value to 7.4 leads to a desaggregation of echinocytes. It was found that picric acid does not induce the aggregation of cells fixed by glutaraldehyde. A substantial decrease in the aggegation of spheric erythrocytes obtained after heating the cells at 50 degrees C was observed.

Cell Aggregation-induced FGF8 Elevation Is Essential for P19 Cell Neural Differentiation

PubMed Central

Wang, Chen; Xia, Caihong; Bian, Wei; Liu, Li; Lin, Wei; Chen, Ye-Guang; Ang, Siew-Lan

2006-01-01

FGF8, a member of the fibroblast growth factor (FGF) family, has been shown to play important roles in different developing systems. Mouse embryonic carcinoma P19 cells could be induced by retinoic acid (RA) to differentiate into neuroectodermal cell lineages, and this process is cell aggregation dependent. In this report, we show that FGF8 expression is transiently up-regulated upon P19 cell aggregation, and the aggregation-dependent FGF8 elevation is pluripotent stem cell related. Overexpressing FGF8 promotes RA-induced monolayer P19 cell neural differentiation. Inhibition of FGF8 expression by RNA interference or blocking FGF signaling by the FGF receptor inhibitor, SU5402, attenuates neural differentiation of the P19 cell. Blocking the bone morphogenetic protein (BMP) pathway by overexpressing Smad6 in P19 cells, we also show that FGF signaling plays a BMP inhibition–independent role in P19 cell neural differentiation. PMID:16641368

Evaluation of Aggregated Ag85B Antigen for Its Biophysical Properties, Immunogenicity, and Vaccination Potential in a Murine Model of Tuberculosis Infection

PubMed Central

Ahmad, Faraz; Zubair, Swaleha; Gupta, Pushpa; Gupta, Umesh Datta; Patel, Rakesh; Owais, Mohammad

2017-01-01

Protein aggregates have been reported to act as a reservoir that can release biologically active, native form of precursor protein. Keeping this fact into consideration, it is tempting to exploit protein aggregate-based antigen delivery system as a functional vaccine to expand desirable immunological response in the host. Herein, we explored the capacity of aggregated Ag85B of Mycobacterium tuberculosis (Mtb) to act as a prophylactic vaccine system that releases the precursor antigen in slow and sustained manner. Being particulate system with exposed hydrophobic residues, aggregated Ag85B is likely to be avidly taken up by both phagocytosis as well as fusion with plasma membrane of antigen presenting cells, leading to its direct delivery to their cytosol. Its unique ability to access cytosol of target cells is further evident from the fact that immunization with aggregated Ag85B led to the induction of Th1-dominant immune response along with upregulated expression of qualitatively superior polyfunctional T cells in the mice. Antibodies generated following immunization with aggregated antigen recognized both native and monomeric Ag85B released from protein aggregate. The implicated immunization strategy offers protection at par to that of established BCG vaccine with desirable central and effector memory responses against subsequent Mtb aerosol challenge. The study highlights the potential of aggregated Ag85B as promising antigen delivery system and paves the way to design better prophylactic regimes against various intracellular pathogens including Mtb. PMID:29230211

A discrete cell model with adaptive signalling for aggregation of Dictyostelium discoideum.

PubMed Central

Dallon, J C; Othmer, H G

1997-01-01

Dictyostelium discoideum (Dd) is a widely studied model system from which fundamental insights into cell movement, chemotaxis, aggregation and pattern formation can be gained. In this system aggregation results from the chemotactic response by dispersed amoebae to a travelling wave of the chemoattractant cAMP. We have developed a model in which the cells are treated as discrete points in a continuum field of the chemoattractant, and transduction of the extracellular cAMP signal into the intracellular signal is based on the G protein model developed by Tang & Othmer. The model reproduces a number of experimental observations and gives further insight into the aggregation process. We investigate different rules for cell movement the factors that influence stream formation the effect on aggregation of noise in the choice of the direction of movement and when spiral waves of chemoattractant and cell density are likely to occur. Our results give new insight into the origin of spiral waves and suggest that streaming is due to a finite amplitude instability. PMID:9134569

Electrophoretic interactions and aggregation of colloidal biological particles

NASA Technical Reports Server (NTRS)

Davis, Robert H.; Nichols, Scott C.; Loewenberg, Michael; Todd, Paul

1994-01-01

The separation of cells or particles from solution has traditionally been accomplished with centrifuges or by sedimentation; however, many particles have specific densities close to unity, making buoyancy-driven motion slow or negligible, but most cells and particles carry surface charges, making them ideal for electrophoretic separation. Both buoyancy-driven and electrophoretic separation may be influenced by hydrodynamic interactions and aggregation of neighboring particles. Aggregation by electrophoresis was analyzed for two non-Brownian particles with different zeta potentials and thin double layers migrating through a viscous fluid. The results indicate that the initial rate of electrophoretically-driven aggregation may exceed that of buoyancy-driven aggregation, even under conditions in which buoyancy-driven relative motion of noninteracting particles is dominant.

Modelling the collective response of heterogeneous cell populations to stationary gradients and chemical signal relay

NASA Astrophysics Data System (ADS)

Pineda, M.; Eftimie, R.

2017-12-01

The directed motion of cell aggregates toward a chemical source occurs in many relevant biological processes. Understanding the mechanisms that control this complex behavior is of great relevance for our understanding of developmental biological processes and many diseases. In this paper, we consider a self-propelled particle model for the movement of heterogeneous subpopulations of chemically interacting cells towards an imposed stable chemical gradient. Our simulations show explicitly how self-organisation of cell populations (which could lead to engulfment or complete cell segregation) can arise from the heterogeneity of chemotactic responses alone. This new result complements current theoretical and experimental studies that emphasise the role of differential cell-cell adhesion on self-organisation and spatial structure of cellular aggregates. We also investigate how the speed of individual cell aggregations increases with the chemotactic sensitivity of the cells, and decreases with the number of cells inside the aggregates

Hsp42 is required for sequestration of protein aggregates into deposition sites in Saccharomyces cerevisiae

PubMed Central

Specht, Sebastian; Miller, Stephanie B.M.

2011-01-01

The aggregation of proteins inside cells is an organized process with cytoprotective function. In Saccharomyces cerevisiae, aggregating proteins are spatially sequestered to either juxtanuclear or peripheral sites, which target distinct quality control pathways for refolding and degradation. The cellular machinery driving the sequestration of misfolded proteins to these sites is unknown. In this paper, we show that one of the two small heat shock proteins of yeast, Hsp42, is essential for the formation of peripheral aggregates during physiological heat stress. Hsp42 preferentially localizes to peripheral aggregates but is largely absent from juxtanuclear aggregates, which still form in hsp42Δ cells. Transferring the amino-terminal domain of Hsp42 to Hsp26, which does not participate in aggregate sorting, enables Hsp26 to replace Hsp42 function. Our data suggest that Hsp42 acts via its amino-terminal domain to coaggregate with misfolded proteins and perhaps link such complexes to further sorting factors. PMID:22065637

The number and growth pattern of plasmacytoid dendritic cells vary in different types of reactive lymph nodes: an immunohistochemical study.

PubMed

Rollins-Raval, Marian A; Marafioti, Teresa; Swerdlow, Steven H; Roth, Christine G

2013-06-01

Plasmacytoid dendritic cells, which play a fundamental role in the innate immune response, are best known for their presence in hyaline-vascular Castleman disease and histiocytic necrotizing lymphadenitis. The relative number and distribution in many reactive entities as detected using more sensitive methods are uncertain, and their diagnostic implications are unknown. Immunohistochemical studies for plasmacytoid dendritic cell-associated markers CD123 and CD2AP were performed on 42 lymph nodes with hyaline-vascular Castleman disease, histiocytic necrotizing lymphadenitis, sarcoidosis, necrotizing granulomatous inflammation, viral infection, dermatopathic lymphadenopathy, autoimmune disease, and a histologic pattern compatible with toxoplasmosis. The overall plasmacytoid dendritic cell numbers and growth patterns (tight aggregates, loose aggregates/clusters, scattered single cells) were assessed. Plasmacytoid dendritic cells were present in all cases and were predominantly distributed in loose aggregates/clusters or singly. They were most numerous in granulomatous inflammation and histiocytic necrotizing lymphadenitis, whereas viral infections showed the fewest overall numbers and a predominant pattern of scattered single cells. Tight aggregates of plasmacytoid dendritic cells were most numerous in hyaline-vascular Castleman disease (100% sensitive, 68% specific). Plasmacytoid dendritic cells are not limited to a small number of reactive lymphadenopathies but are found in many reactive processes, often with a predominant pattern of loose aggregates/clusters and scattered single cells. However, tight aggregates were a characteristic feature of hyaline-vascular Castleman disease, and viral infections typically showed only few scattered cells distributed singly. Copyright © 2013 Elsevier Inc. All rights reserved.

Why are enteric ganglia so small? Role of differential adhesion of enteric neurons and enteric neural crest cells.

PubMed Central

Rollo, Benjamin N.; Zhang, Dongcheng; Simkin, Johanna E.; Menheniott, Trevelyan R.; Newgreen, Donald F.

2015-01-01

The avian enteric nervous system (ENS) consists of a vast number of unusually small ganglia compared to other peripheral ganglia. Each ENS ganglion at mid-gestation has a core of neurons and a shell of mesenchymal precursor/glia-like enteric neural crest (ENC) cells. To study ENS cell ganglionation we isolated midgut ENS cells by HNK-1 fluorescence-activated cell sorting (FACS) from E5 and E8 quail embryos, and from E9 chick embryos. We performed cell-cell aggregation assays which revealed a developmentally regulated functional increase in ENS cell adhesive function, requiring both Ca 2+ -dependent and independent adhesion. This was consistent with N-cadherin and NCAM labelling. Neurons sorted to the core of aggregates, surrounded by outer ENC cells, showing that neurons had higher adhesion than ENC cells. The outer surface of aggregates became relatively non-adhesive, correlating with low levels of NCAM and N-cadherin on this surface of the outer non-neuronal ENC cells. Aggregation assays showed that ENS cells FACS selected for NCAM-high and enriched for enteric neurons formed larger and more coherent aggregates than unsorted ENS cells. In contrast, ENS cells of the NCAM-low FACS fraction formed small, disorganised aggregates.  This suggests a novel mechanism for control of ENS ganglion morphogenesis where i) differential adhesion of ENS neurons and ENC cells controls the core/shell ganglionic structure and ii) the ratio of neurons to ENC cells dictates the equilibrium ganglion size by generation of an outer non-adhesive surface. PMID:26064478

The effect of protein acetylation on the formation and processing of inclusion bodies and endogenous protein aggregates in Escherichia coli cells.

PubMed

Kuczyńska-Wiśnik, Dorota; Moruno-Algara, María; Stojowska-Swędrzyńska, Karolina; Laskowska, Ewa

2016-11-10

Acetylation of lysine residues is a reversible post-translational modification conserved from bacteria to humans. Several recent studies have revealed hundreds of lysine-acetylated proteins in various bacteria; however, the physiological role of these modifications remains largely unknown. Since lysine acetylation changes the size and charge of proteins and thereby may affect their conformation, we assumed that lysine acetylation can stimulate aggregation of proteins, especially for overproduced recombinant proteins that form inclusion bodies. To verify this assumption, we used Escherichia coli strains that overproduce aggregation-prone VP1GFP protein. We found that in ΔackA-pta cells, which display diminished protein acetylation, inclusion bodies were formed with a delay and processed faster than in the wild-type cells. Moreover, in ΔackA-pta cells, inclusion bodies exhibited significantly increased specific GFP fluorescence. In CobB deacetylase-deficient cells, in which protein acetylation was enhanced, the formation of inclusion bodies was increased and their processing was significantly inhibited. Similar results were obtained with regard to endogenous protein aggregates formed during the late stationary phase in ΔackA-pta and ΔcobB cells. Our studies revealed that protein acetylation affected the aggregation of endogenous E. coli proteins and the yield, solubility, and biological activity of a model recombinant protein. In general, decreased lysine acetylation inhibited the formation of protein aggregates, whereas increased lysine acetylation stabilized protein aggregates. These findings should be considered during the designing of efficient strategies for the production of recombinant proteins in E. coli cells.

Uncoupling of Protein Aggregation and Neurodegeneration in a Mouse Amyotrophic Lateral Sclerosis Model.

PubMed

Lee, Joo-Yong; Kawaguchi, Yoshiharu; Li, Ming; Kapur, Meghan; Choi, Su Jin; Kim, Hak-June; Park, Song-Yi; Zhu, Haining; Yao, Tso-Pang

2015-01-01

Aberrant accumulation of protein aggregates is a pathological hallmark of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Although a buildup of protein aggregates frequently leads to cell death, whether it is the key pathogenic factor in driving neurodegenerative disease remains controversial. HDAC6, a cytosolic ubiquitin-binding deacetylase, has emerged as an important regulator of ubiquitin-dependent quality control autophagy, a lysosome-dependent degradative system responsible for the disposal of misfolded protein aggregates and damaged organelles. Here, we show that in cell models HDAC6 plays a protective role against multiple disease-associated and aggregation-prone cytosolic proteins by facilitating their degradation. We further show that HDAC6 is required for efficient localization of lysosomes to protein aggregates, indicating that lysosome targeting to autophagic substrates is regulated. Supporting a critical role of HDAC6 in protein aggregate disposal in vivo, genetic ablation of HDAC6 in a transgenic SOD1G93A mouse, a model of ALS, leads to dramatic accumulation of ubiquitinated SOD1G93A protein aggregates. Surprisingly, despite a robust buildup of SOD1G93A aggregates, deletion of HDAC6 only moderately modified the motor phenotypes. These findings indicate that SOD1G93A aggregation is not the only determining factor to drive neurodegeneration in ALS, and that HDAC6 likely modulates neurodegeneration through additional mechanisms beyond protein aggregate clearance. © 2015 S. Karger AG, Basel.

Aggregate complexes of HIV-1 induced by multimeric antibodies.

PubMed

Stieh, Daniel J; King, Deborah F; Klein, Katja; Liu, Pinghuang; Shen, Xiaoying; Hwang, Kwan Ki; Ferrari, Guido; Montefiori, David C; Haynes, Barton; Pitisuttithum, Punnee; Kaewkungwal, Jaranit; Nitayaphan, Sorachai; Rerks-Ngarm, Supachai; Michael, Nelson L; Robb, Merlin L; Kim, Jerome H; Denny, Thomas N; Tomaras, Georgia D; Shattock, Robin J

2014-10-02

Antibody mediated viral aggregation may impede viral transfer across mucosal surfaces by hindering viral movement in mucus, preventing transcytosis, or reducing inter-cellular penetration of epithelia thereby limiting access to susceptible mucosal CD4 T cells and dendritic cells. These functions may work together to provide effective immune exclusion of virus from mucosal tissue; however little is known about the antibody characteristics required to induce HIV aggregation. Such knowledge may be critical to the design of successful immunization strategies to facilitate viral immune exclusion at the mucosal portals of entry. The potential of neutralizing and non-neutralizing IgG and IgA monoclonals (mAbs) to induce HIV-1 aggregation was assessed by Dynamic light scattering (DLS). Although neutralizing and non-neutralizing IgG mAbs and polyclonal HIV-Ig efficiently aggregated soluble Env trimers, they were not capable of forming viral aggregates. In contrast, dimeric (but not monomeric) IgA mAbs induced stable viral aggregate populations that could be separated from uncomplexed virions. Epitope specificity influenced both the degree of aggregation and formation of higher order complexes by dIgA. IgA purified from serum of uninfected RV144 vaccine trial responders were able to efficiently opsonize viral particles in the absence of significant aggregation, reflective of monomeric IgA. These results collectively demonstrate that dIgA is capable of forming stable viral aggregates providing a plausible basis for testing the effectiveness of aggregation as a potential protection mechanism at the mucosal portals of viral entry.

Quantifying Aggregation Dynamics during Myxococcus xanthus Development▿†

PubMed Central

Zhang, Haiyang; Angus, Stuart; Tran, Michael; Xie, Chunyan; Igoshin, Oleg A.; Welch, Roy D.

2011-01-01

Under starvation conditions, a swarm of Myxococcus xanthus cells will undergo development, a multicellular process culminating in the formation of many aggregates called fruiting bodies, each of which contains up to 100,000 spores. The mechanics of symmetry breaking and the self-organization of cells into fruiting bodies is an active area of research. Here we use microcinematography and automated image processing to quantify several transient features of developmental dynamics. An analysis of experimental data indicates that aggregation reaches its steady state in a highly nonmonotonic fashion. The number of aggregates rapidly peaks at a value 2- to 3-fold higher than the final value and then decreases before reaching a steady state. The time dependence of aggregate size is also nonmonotonic, but to a lesser extent: average aggregate size increases from the onset of aggregation to between 10 and 15 h and then gradually decreases thereafter. During this process, the distribution of aggregates transitions from a nearly random state early in development to a more ordered state later in development. A comparison of experimental results to a mathematical model based on the traffic jam hypothesis indicates that the model fails to reproduce these dynamic features of aggregation, even though it accurately describes its final outcome. The dynamic features of M. xanthus aggregation uncovered in this study impose severe constraints on its underlying mechanisms. PMID:21784940

Morphology-Variable Aggregates Prepared from Cholesterol-Containing Amphiphilic Glycopolymers: Their Protein Recognition/Adsorption and Drug Delivery Applications

PubMed Central

Wang, Zhao; Luo, Ting; Cao, Amin; Sun, Jingjing; Jia, Lin

2018-01-01

In this study, a series of diblock glycopolymers, poly(6-O-methacryloyl-d-galactopyranose)-b-poly(6-cholesteryloxyhexyl methacrylate) (PMAgala-b-PMAChols), with cholesterol/galactose grafts were prepared through a sequential reversible addition-fragmentation chain transfer (RAFT) polymerization and deprotection process. The glycopolymers could self-assemble into aggregates with various morphologies depending on cholesterol/galactose-containing block weight ratios, as determined by transmission electronic microscopy (TEM) and dynamic laser light scattering (DLS). In addition, the lectin (Ricinus communis agglutinin II, RCA120) recognition and bovine serum albumin (BSA) adsorption of the PMAgala-b-PMAChol aggregates were evaluated. The SK-Hep-1 tumor cell inhibition properties of the PMAgala-b-PMAChol/doxorubicin (DOX) complex aggregates were further examined in vitro. Results indicate that the PMAgala-b-PMAChol aggregates with various morphologies showed different interaction/recognition features with RCA120 and BSA. Spherical aggregates (d ≈ 92 nm) possessed the highest RCA120 recognition ability and lowest BSA protein adsorption. In addition, the DOX-loaded spherical complex aggregates exhibited a better tumor cell inhibition property than those of nanofibrous complex aggregates. The morphology-variable aggregates derived from the amphiphilic glycopolymers may serve as multifunctional biomaterials with biomolecular recognition and drug delivery features. PMID:29495614

Proteins with Intrinsically Disordered Domains Are Preferentially Recruited to Polyglutamine Aggregates

PubMed Central

O’Meally, Robert; Sonnenberg, Jason L.; Cole, Robert N.; Shewmaker, Frank P.

2015-01-01

Intracellular protein aggregation is the hallmark of several neurodegenerative diseases. Aggregates formed by polyglutamine (polyQ)-expanded proteins, such as Huntingtin, adopt amyloid-like structures that are resistant to denaturation. We used a novel purification strategy to isolate aggregates formed by human Huntingtin N-terminal fragments with expanded polyQ tracts from both yeast and mammalian (PC-12) cells. Using mass spectrometry we identified the protein species that are trapped within these polyQ aggregates. We found that proteins with very long intrinsically-disordered (ID) domains (≥100 amino acids) and RNA-binding proteins were disproportionately recruited into aggregates. The removal of the ID domains from selected proteins was sufficient to eliminate their recruitment into polyQ aggregates. We also observed that several neurodegenerative disease-linked proteins were reproducibly trapped within the polyQ aggregates purified from mammalian cells. Many of these proteins have large ID domains and are found in neuronal inclusions in their respective diseases. Our study indicates that neurodegenerative disease-associated proteins are particularly vulnerable to recruitment into polyQ aggregates via their ID domains. Also, the high frequency of ID domains in RNA-binding proteins may explain why RNA-binding proteins are frequently found in pathological inclusions in various neurodegenerative diseases. PMID:26317359

The role of synaptotagmin I C2A calcium-binding domain in synaptic vesicle clustering during synapse formation

PubMed Central

Gardzinski, Peter; Lee, David W K; Fei, Guang-He; Hui, Kwokyin; Huang, Guan J; Sun, Hong-Shuo; Feng, Zhong-Ping

2007-01-01

Synaptic vesicles aggregate at the presynaptic terminal during synapse formation via mechanisms that are poorly understood. Here we have investigated the role of the putative calcium sensor synaptotagmin I in vesicle aggregation during the formation of soma–soma synapses between identified partner cells using a simple in vitro synapse model in the mollusc Lymnaea stagnalis. Immunocytochemistry, optical imaging and electrophysiological recording techniques were used to monitor synapse formation and vesicle localization. Within 6 h, contact between appropriate synaptic partner cells up-regulated global synaptotagmin I expression, and induced a localized aggregation of synaptotagmin I at the contact site. Cell contacts between non-synaptic partner cells did not affect synaptotagmin I expression. Application of an human immunodeficiency virus type-1 transactivator (HIV-1 TAT)-tagged peptide corresponding to loop 3 of the synaptotagmin I C2A domain prevented synaptic vesicle aggregation and synapse formation. By contrast, a TAT-tagged peptide containing the calcium-binding motif of the C2B domain did not affect synaptic vesicle aggregation or synapse formation. Calcium imaging with Fura-2 demonstrated that TAT–C2 peptides did not alter either basal or evoked intracellular calcium levels. These results demonstrate that contact with an appropriate target cell is necessary to initiate synaptic vesicle aggregation during nascent synapse formation and that the initial aggregation of synaptic vesicles is dependent on loop 3 of the C2A domain of synaptotagmin I. PMID:17317745

Diatom aggregation and dimethylsulfide production in phytoplankton blooms

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

Crocker, K.M.

1994-01-01

Phytoplankton blooms are crucial links in many of the earth's biogeochemical cycles. Blooms take up atmospheric carbon through photosynthesis, and sequester it on the ocean floor by sinking. Aggregation of single cells into [open quote]marine snow[close quote] particles speeds up the sinking of algal cells. Laboratory studies investigating the process of aggregation show that some species have a higher probability of aggregating than others, and that there exist several mechanisms for causing aggregation. Field studies confirm that some species are more likely to be found in aggregates than in the surrounding seawater. High latitude Premnesiophyte blooms are found to producemore » large amounts of dimethylsulflde (DMS), believed to be an important chemical in global thermoregulation. DMS is found to vary diurnally, possibly due to photooxidation by ultraviolet light. This possibility links the effects of DMS on cloud formation with the effects of increased ultraviolet light penetrating the earths ozone layer.« less

Effect of the LHCII pigment-protein complex aggregation on photovoltaic properties of sensitized TiO2 solar cells.

PubMed

Yang, Yiqun; Jankowiak, Ryszard; Lin, Chen; Pawlak, Krzysztof; Reus, Michael; Holzwarth, Alfred R; Li, Jun

2014-10-14

A modified dye-sensitized solar cell consisting of a thin TiO2 barrier layer sensitized with natural trimeric light-harvesting complex II (LHCII) from spinach was used as a biomimetic model to study the effects of LHCII aggregation on the photovoltaic properties. The aggregation of individual trimers induced molecular reorganization, which dramatically increased the photocurrent. The morphology of small- and large-size LHCII aggregates deposited on a surface was confirmed by atomic force microscopy. Enhanced LHCII immobilization was accomplished via electrostatic interaction with amine-functionalized photoanodes. The photocurrent responses of the assembled solar cells under illumination at three characteristic wavelength bands in the UV-Vis absorption spectra of LHCII solutions confirmed that a significant photocurrent was generated by LHCII photosensitizers. The enhanced photocurrent by large aggregated LHCII is shown to correlate with the quenching in the far-red fluorescence deriving from chlorophyll-chlorophyll charge transfer states that are effectively coupled with the TiO2 surface and thus inject electrons into the TiO2 conduction band. The large aggregated LHCII with more chlorophyll-chlorophyll charge transfer states is a much better sensitizer since it injects electrons more efficiently into the conduction band of TiO2 than the small aggregated LHCII mostly consisting of unquenched chlorophyll excited state. The assembled solar cells demonstrated remarkable stability in both aqueous buffer and acetonitrile electrolytes over 30 days.

Modulation of mutant Huntingtin aggregates and toxicity by human myeloid leukemia factors.

PubMed

Banerjee, Manisha; Datta, Moumita; Bhattacharyya, Nitai P

2017-01-01

Increased poly glutamine (polyQ) stretch at N-terminal of Huntingtin (HTT) causes Huntington's disease. HTT interacts with large number of proteins, although the preference for such interactions with wild type or mutated HTT protein remains largely unknown. HYPK, an intrinsically unstructured protein chaperone and interactor of mutant HTT was found to interact with myeloid leukemia factor 1 (MLF1) and 2 (MLF2). To identify the role of these two proteins in mutant HTT mediated aggregate formation and toxicity in a cell model, both the proteins were found to preferentially interact with the mutated N-terminal HTT. They significantly reduced the number of cells containing mutant HTT aggregates and subsequent apoptosis in Neuro2A cells. Additionally, in FRAP assay, mobile fraction of mutant HTT aggregates was increased in the presence of MLF1 or MLF2. Further, MLF1 could release transcription factors like p53, CBP and CREB from mutant HTT aggregates. Moreover, in HeLa cell co-expressing mutant HTT exon1 and full length MLF1, p53 was released from the aggregates, leading to the recovery of the expression of the GADD45A transcript, a p53 regulated gene. Taking together, these results showed that MLF1 and MLF2 modulated the formation of aggregates and induction of apoptosis as well as the expressions of genes indirectly. Copyright © 2016 Elsevier Ltd. All rights reserved.

nDEP-driven cell patterning and bottom-up construction of cell aggregates using a new bioelectronic chip.

PubMed

Menad, S; Franqueville, L; Haddour, N; Buret, F; Frenea-Robin, M

2015-04-01

Creating cell aggregates of controlled size and shape and patterning cells on substrates using a bottom-up approach constitutes important challenges for tissue-engineering applications and studies of cell-cell interactions. In this paper, we report nDEP (negative dielectrophoresis) driven assembly of cells as compact aggregates or onto defined areas using a new bioelectronic chip. This chip is composed of a quadripolar electrode array obtained using coplanar electrodes partially covered with a thin, micropatterned PDMS membrane. This thin PDMS layer was coated with poly-L-lysine and played the role of adhesive substrate for cell patterning. For the formation of detachable cell aggregates, the PDMS was not pretreated and cells were simply immobilized into assemblies maintained by cell-cell adhesion after the electric field removal. Cell viability after exposition to DEP buffer was also assessed, as well as cell spreading activity following DEP-driven assembly. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Prediction of the aggregation propensity of proteins from the primary sequence: aggregation properties of proteomes.

PubMed

Castillo, Virginia; Graña-Montes, Ricardo; Sabate, Raimon; Ventura, Salvador

2011-06-01

In the cell, protein folding into stable globular conformations is in competition with aggregation into non-functional and usually toxic structures, since the biophysical properties that promote folding also tend to favor intermolecular contacts, leading to the formation of β-sheet-enriched insoluble assemblies. The formation of protein deposits is linked to at least 20 different human disorders, ranging from dementia to diabetes. Furthermore, protein deposition inside cells represents a major obstacle for the biotechnological production of polypeptides. Importantly, the aggregation behavior of polypeptides appears to be strongly influenced by the intrinsic properties encoded in their sequences and specifically by the presence of selective short regions with high aggregation propensity. This allows computational methods to be used to analyze the aggregation properties of proteins without the previous requirement for structural information. Applications range from the identification of individual amyloidogenic regions in disease-linked polypeptides to the analysis of the aggregation properties of complete proteomes. Herein, we review these theoretical approaches and illustrate how they have become important and useful tools in understanding the molecular mechanisms underlying protein aggregation. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Ito, Norie; Department of Neurology, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-ku, Sapporo 060-8556; Kamiguchi, Kenjiro

Polyglutamine (polyQ) diseases comprise neurodegenerative disorders caused by expression of expanded polyQ-containing proteins. The cytotoxicity of the expanded polyQ-containing proteins is closely associated with aggregate formation. In this study, we report that a novel J-protein, DNAJ (HSP40) Homolog, Subfamily C, Member 8 (DNAJC8), suppresses the aggregation of polyQ-containing protein in a cellular model of spinocerebellar ataxia type 3 (SCA3), which is also known as Machado-Joseph disease. Overexpression of DNAJC8 in SH-SY5Y neuroblastoma cells significantly reduced the polyQ aggregation and apoptosis, and DNAJC8 was co-localized with the polyQ aggregation in the cell nucleus. Deletion mutants of DNAJC8 revealed that the C-terminalmore » domain of DNAJC8 was essential for the suppression of polyQ aggregation, whereas the J-domain was dispensable. Furthermore, 22-mer oligopeptide derived from C-termilal domain could suppress the polyQ aggregation. These results indicate that DNAJC8 can suppress the polyQ aggregation via a distinct mechanism independent of HSP70-based chaperone machinery and have a unique protective role against the aggregation of expanded polyQ-containing proteins such as pathogenic ataxin-3 proteins.« less

Cartilage Derived from Bone Marrow Mesenchymal Stem Cells Expresses Lubricin In Vitro and In Vivo

PubMed Central

Nakagawa, Yusuke; Muneta, Takeshi; Otabe, Koji; Ozeki, Nobutake; Mizuno, Mitsuru; Udo, Mio; Saito, Ryusuke; Yanagisawa, Katsuaki; Ichinose, Shizuko; Koga, Hideyuki; Tsuji, Kunikazu; Sekiya, Ichiro

2016-01-01

Objective Lubricin expression in the superficial cartilage will be a crucial factor in the success of cartilage regeneration. Mesenchymal stem cells (MSCs) are an attractive cell source and the use of aggregates of MSCs has some advantages in terms of chondrogenic potential and efficiency of cell adhesion. Lubricin expression in transplanted MSCs has not been fully elucidated so far. Our goals were to determine (1) whether cartilage pellets of human MSCs expressed lubricin in vitro chondrogenesis, (2) whether aggregates of human MSCs promoted lubricin expression, and (3) whether aggregates of MSCs expressed lubricin in the superficial cartilage after transplantation into osteochondral defects in rats. Methods For in vitro analysis, human bone marrow (BM) MSCs were differentiated into cartilage by pellet culture, and also aggregated using the hanging drop technique. For an animal study, aggregates of BM MSCs derived from GFP transgenic rats were transplanted to the osteochondral defect in the trochlear groove of wild type rat knee joints. Lubricin expression was mainly evaluated in differentiated and regenerated cartilages. Results In in vitro analysis, lubricin was detected in the superficial zone of the pellets and conditioned medium. mRNA expression of Proteoglycan4 (Prg4), which encodes lubricin, in pellets was significantly higher than that of undifferentiated MSCs. Aggregates showed different morphological features between the superficial and deep zone, and the Prg4 mRNA expression increased after aggregate formation. Lubricin was also found in the aggregate. In a rat study, articular cartilage regeneration was significantly better in the MSC group than in the control group as shown by macroscopical and histological analysis. The transmission electron microscope showed that morphology of the superficial cartilage in the MSC group was closer to that of the intact cartilage than in the control group. GFP positive cells remained in the repaired tissue and expressed lubricin in the superficial cartilage. Conclusion Cartilage derived from MSCs expressed lubricin protein both in vitro and in vivo. Aggregation promoted lubricin expression of MSCs in vitro and transplantation of aggregates of MSCs regenerated cartilage including the superficial zone in a rat osteochondral defect model. Our results indicate that aggregated MSCs could be clinically relevant for therapeutic approaches to articular cartilage regeneration with an appropriate superficial zone in the future. PMID:26867127

Cartilage Derived from Bone Marrow Mesenchymal Stem Cells Expresses Lubricin In Vitro and In Vivo.

PubMed

Nakagawa, Yusuke; Muneta, Takeshi; Otabe, Koji; Ozeki, Nobutake; Mizuno, Mitsuru; Udo, Mio; Saito, Ryusuke; Yanagisawa, Katsuaki; Ichinose, Shizuko; Koga, Hideyuki; Tsuji, Kunikazu; Sekiya, Ichiro

2016-01-01

Lubricin expression in the superficial cartilage will be a crucial factor in the success of cartilage regeneration. Mesenchymal stem cells (MSCs) are an attractive cell source and the use of aggregates of MSCs has some advantages in terms of chondrogenic potential and efficiency of cell adhesion. Lubricin expression in transplanted MSCs has not been fully elucidated so far. Our goals were to determine (1) whether cartilage pellets of human MSCs expressed lubricin in vitro chondrogenesis, (2) whether aggregates of human MSCs promoted lubricin expression, and (3) whether aggregates of MSCs expressed lubricin in the superficial cartilage after transplantation into osteochondral defects in rats. For in vitro analysis, human bone marrow (BM) MSCs were differentiated into cartilage by pellet culture, and also aggregated using the hanging drop technique. For an animal study, aggregates of BM MSCs derived from GFP transgenic rats were transplanted to the osteochondral defect in the trochlear groove of wild type rat knee joints. Lubricin expression was mainly evaluated in differentiated and regenerated cartilages. In in vitro analysis, lubricin was detected in the superficial zone of the pellets and conditioned medium. mRNA expression of Proteoglycan4 (Prg4), which encodes lubricin, in pellets was significantly higher than that of undifferentiated MSCs. Aggregates showed different morphological features between the superficial and deep zone, and the Prg4 mRNA expression increased after aggregate formation. Lubricin was also found in the aggregate. In a rat study, articular cartilage regeneration was significantly better in the MSC group than in the control group as shown by macroscopical and histological analysis. The transmission electron microscope showed that morphology of the superficial cartilage in the MSC group was closer to that of the intact cartilage than in the control group. GFP positive cells remained in the repaired tissue and expressed lubricin in the superficial cartilage. Cartilage derived from MSCs expressed lubricin protein both in vitro and in vivo. Aggregation promoted lubricin expression of MSCs in vitro and transplantation of aggregates of MSCs regenerated cartilage including the superficial zone in a rat osteochondral defect model. Our results indicate that aggregated MSCs could be clinically relevant for therapeutic approaches to articular cartilage regeneration with an appropriate superficial zone in the future.

Analysis and modification of defective surface aggregates on PCDTBT:PCBM solar cell blends using combined Kelvin probe, conductive and bimodal atomic force microscopy

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

Noh, Hanaul; Diaz, Alfredo J.; Solares, Santiago D.

Organic photovoltaic systems comprising donor polymers and acceptor fullerene derivatives are attractive for inexpensive energy harvesting. Extensive research on polymer solar cells has provided insight into the factors governing device-level efficiency and stability. However, the detailed investigation of nanoscale structures is still challenging. Here we demonstrate the analysis and modification of unidentified surface aggregates. The aggregates are characterized electrically by Kelvin probe force microscopy and conductive atomic force microscopy (C-AFM), whereby the correlation between local electrical potential and current confirms a defective charge transport. Bimodal AFM modification confirms that the aggregates exist on top of the solar cell structure, andmore » is used to remove them and to reveal the underlying active layer. The systematic analysis of the surface aggregates suggests that the structure consists of PCBM molecules.« less

Analysis and modification of defective surface aggregates on PCDTBT:PCBM solar cell blends using combined Kelvin probe, conductive and bimodal atomic force microscopy

DOE PAGES

Noh, Hanaul; Diaz, Alfredo J.; Solares, Santiago D.

2017-03-08

Organic photovoltaic systems comprising donor polymers and acceptor fullerene derivatives are attractive for inexpensive energy harvesting. Extensive research on polymer solar cells has provided insight into the factors governing device-level efficiency and stability. However, the detailed investigation of nanoscale structures is still challenging. Here we demonstrate the analysis and modification of unidentified surface aggregates. The aggregates are characterized electrically by Kelvin probe force microscopy and conductive atomic force microscopy (C-AFM), whereby the correlation between local electrical potential and current confirms a defective charge transport. Bimodal AFM modification confirms that the aggregates exist on top of the solar cell structure, andmore » is used to remove them and to reveal the underlying active layer. The systematic analysis of the surface aggregates suggests that the structure consists of PCBM molecules.« less

Analysis and modification of defective surface aggregates on PCDTBT:PCBM solar cell blends using combined Kelvin probe, conductive and bimodal atomic force microscopy

PubMed Central

Noh, Hanaul; Diaz, Alfredo J

2017-01-01

Organic photovoltaic systems comprising donor polymers and acceptor fullerene derivatives are attractive for inexpensive energy harvesting. Extensive research on polymer solar cells has provided insight into the factors governing device-level efficiency and stability. However, the detailed investigation of nanoscale structures is still challenging. Here we demonstrate the analysis and modification of unidentified surface aggregates. The aggregates are characterized electrically by Kelvin probe force microscopy and conductive atomic force microscopy (C-AFM), whereby the correlation between local electrical potential and current confirms a defective charge transport. Bimodal AFM modification confirms that the aggregates exist on top of the solar cell structure, and is used to remove them and to reveal the underlying active layer. The systematic analysis of the surface aggregates suggests that the structure consists of PCBM molecules. PMID:28382247

Lewy Body-like α-Synuclein Aggregates Resist Degradation and Impair Macroautophagy*♦

PubMed Central

Tanik, Selcuk A.; Schultheiss, Christine E.; Volpicelli-Daley, Laura A.; Brunden, Kurt R.; Lee, Virginia M. Y.

2013-01-01

Cytoplasmic α-synuclein (α-syn) aggregates, referred to as Lewy bodies, are pathological hallmarks of a number of neurodegenerative diseases, most notably Parkinson disease. Activation of macroautophagy is suggested to facilitate degradation of certain proteinaceous inclusions, but it is unclear if this pathway is capable of degrading α-syn aggregates. Here, we examined this issue by utilizing cellular models in which intracellular Lewy body-like α-syn inclusions accumulate after internalization of pre-formed α-syn fibrils into α-syn-expressing HEK293 cells or cultured primary neurons. We demonstrate that α-syn inclusions cannot be effectively degraded, even though they co-localize with essential components of both the autophagic and proteasomal protein degradation pathways. The α-syn aggregates persist even after soluble α-syn levels have been substantially reduced, suggesting that once formed, the α-syn inclusions are refractory to clearance. Importantly, we also find that α-syn aggregates impair overall macroautophagy by reducing autophagosome clearance, which may contribute to the increased cell death that is observed in aggregate-bearing cells. PMID:23532841

α-Synuclein and huntingtin exon 1 amyloid fibrils bind laterally to the cellular membrane.

PubMed

Monsellier, Elodie; Bousset, Luc; Melki, Ronald

2016-01-13

Fibrillar aggregates involved in neurodegenerative diseases have the ability to spread from one cell to another in a prion-like manner. The underlying molecular mechanisms, in particular the binding mode of the fibrils to cell membranes, are poorly understood. In this work we decipher the modality by which aggregates bind to the cellular membrane, one of the obligatory steps of the propagation cycle. By characterizing the binding properties of aggregates made of α-synuclein or huntingtin exon 1 protein displaying similar composition and structure but different lengths to mammalian cells we demonstrate that in both cases aggregates bind laterally to the cellular membrane, with aggregates extremities displaying little or no role in membrane binding. Lateral binding to artificial liposomes was also observed by transmission electron microscopy. In addition we show that although α-synuclein and huntingtin exon 1 fibrils bind both laterally to the cellular membrane, their mechanisms of interaction differ. Our findings have important implications for the development of future therapeutic tools that aim to block protein aggregates propagation in the brain.

α-Synuclein and huntingtin exon 1 amyloid fibrils bind laterally to the cellular membrane

PubMed Central

Monsellier, Elodie; Bousset, Luc; Melki, Ronald

2016-01-01

Fibrillar aggregates involved in neurodegenerative diseases have the ability to spread from one cell to another in a prion-like manner. The underlying molecular mechanisms, in particular the binding mode of the fibrils to cell membranes, are poorly understood. In this work we decipher the modality by which aggregates bind to the cellular membrane, one of the obligatory steps of the propagation cycle. By characterizing the binding properties of aggregates made of α-synuclein or huntingtin exon 1 protein displaying similar composition and structure but different lengths to mammalian cells we demonstrate that in both cases aggregates bind laterally to the cellular membrane, with aggregates extremities displaying little or no role in membrane binding. Lateral binding to artificial liposomes was also observed by transmission electron microscopy. In addition we show that although α-synuclein and huntingtin exon 1 fibrils bind both laterally to the cellular membrane, their mechanisms of interaction differ. Our findings have important implications for the development of future therapeutic tools that aim to block protein aggregates propagation in the brain. PMID:26757959

Enhanced Biological Functions of Human Mesenchymal Stem-Cell Aggregates Incorporating E-Cadherin-Modified PLGA Microparticles.

PubMed

Zhang, Yan; Mao, Hongli; Gao, Chao; Li, Suhua; Shuai, Qizhi; Xu, Jianbin; Xu, Ke; Cao, Lei; Lang, Ren; Gu, Zhongwei; Akaike, Toshihiro; Yang, Jun

2016-08-01

Mesenchymal stem cells (MSCs) have emerged as a promising source of multipotent cells for various cell-based therapies due to their unique properties, and formation of 3D MSC aggregates has been explored as a potential strategy to enhance therapeutic efficacy. In this study, poly(lactic-co-glycolic acid) (PLGA) microparticles modified with human E-cadherin fusion protein (hE-cad-PLGA microparticles) have been fabricated and integrated with human MSCs to form 3D cell aggregates. The results show that, compared with the plain PLGA, the hE-cad-PLGA microparticles distribute within the aggregates more evenly and further result in a more significant improvement of cellular proliferation and secretion of a series of bioactive factors due to the synergistic effects from the bioactive E-cadherin fragments and the PLGA microparticles. Meanwhile, the hE-cad-PLGA microparticles incorporated in the aggregates upregulate the phosphorylation of epidermal growth factor receptors and activate the AKT and ERK1/2 signaling pathways in the MSCs. Additionally, the E-cadherin/β-catenin cellular membrane complex in the MSCs is markedly stimulated by the hE-cad-PLGA microparticles. Therefore, engineering 3D cell aggregates with hE-cad-PLGA microparticles can be a promising method for ex vivo multipotent stem-cell expansion with enhanced biological functions and may offer a novel route to expand multipotent stem-cell-based clinical applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Prions on the run: How extracellular vesicles serve as delivery vehicles for self-templating protein aggregates.

PubMed

Liu, Shu; Hossinger, André; Göbbels, Sarah; Vorberg, Ina M

2017-03-04

Extracellular vesicles (EVs) are actively secreted, membrane-bound communication vehicles that exchange biomolecules between cells. EVs also serve as dissemination vehicles for pathogens, including prions, proteinaceous infectious agents that cause transmissible spongiform encephalopathies (TSEs) in mammals. Increasing evidence accumulates that diverse protein aggregates associated with common neurodegenerative diseases are packaged into EVs as well. Vesicle-mediated intercellular transmission of protein aggregates can induce aggregation of homotypic proteins in acceptor cells and might thereby contribute to disease progression. Our knowledge of how protein aggregates are sorted into EVs and how these vesicles adhere to and fuse with target cells is limited. Here we review how TSE prions exploit EVs for intercellular transmission and compare this to the transmission behavior of self-templating cytosolic protein aggregates derived from the yeast prion domain Sup 35 NM. Artificial NM prions are non-toxic to mammalian cell cultures and do not cause loss-of-function phenotypes. Importantly, NM particles are also secreted in association with exosomes that horizontally transmit the prion phenotype to naive bystander cells, a process that can be monitored with high accuracy by automated high throughput confocal microscopy. The high abundance of mammalian proteins with amino acid stretches compositionally similar to yeast prion domains makes the NM cell model an attractive model to study self-templating and dissemination properties of proteins with prion-like domains in the mammalian context.

Multilevel Space-Time Aggregation for Bright Field Cell Microscopy Segmentation and Tracking

PubMed Central

Inglis, Tiffany; De Sterck, Hans; Sanders, Geoffrey; Djambazian, Haig; Sladek, Robert; Sundararajan, Saravanan; Hudson, Thomas J.

2010-01-01

A multilevel aggregation method is applied to the problem of segmenting live cell bright field microscope images. The method employed is a variant of the so-called “Segmentation by Weighted Aggregation” technique, which itself is based on Algebraic Multigrid methods. The variant of the method used is described in detail, and it is explained how it is tailored to the application at hand. In particular, a new scale-invariant “saliency measure” is proposed for deciding when aggregates of pixels constitute salient segments that should not be grouped further. It is shown how segmentation based on multilevel intensity similarity alone does not lead to satisfactory results for bright field cells. However, the addition of multilevel intensity variance (as a measure of texture) to the feature vector of each aggregate leads to correct cell segmentation. Preliminary results are presented for applying the multilevel aggregation algorithm in space time to temporal sequences of microscope images, with the goal of obtaining space-time segments (“object tunnels”) that track individual cells. The advantages and drawbacks of the space-time aggregation approach for segmentation and tracking of live cells in sequences of bright field microscope images are presented, along with a discussion on how this approach may be used in the future work as a building block in a complete and robust segmentation and tracking system. PMID:20467468

Incorporation of Biomaterials in Multicellular Aggregates Modulates Pluripotent Stem Cell Differentiation

PubMed Central

Bratt-Leal, Andrés M.; Carpenedo, Richard L.; Ungrin, Mark; Zandstra, Peter W.; McDevitt, Todd C.

2010-01-01

Biomaterials are increasingly being used to engineer the biochemical and biophysical properties of the extracellular stem cell microenvironment in order to tailor niche characteristics and direct cell phenotype. To date, stem cell-biomaterial interactions have largely been studied by introducing stem cells into artificial environments, such as 2D cell culture on biomaterial surfaces, encapsulation of cell suspensions within hydrogel materials, or cell seeding on 3D polymeric scaffolds. In this study, microparticles fabricated from different materials, such as agarose, PLGA and gelatin, were stably integrated, in a dose-dependent manner, within aggregates of pluripotent stem cells (PSCs) prior to differentiation as a means to directly examine stem cell-biomaterial interactions in 3D. Interestingly, the presence of the materials within the stem cell aggregates differentially modulated the gene and protein expression patterns of several differentiation markers without adversely affecting cell viability. Microparticle incorporation within 3D stem cell aggregates can control the spatial presentation of extracellular environmental cues (i.e. soluble factors, extracellular matrix and intercellular adhesion molecules) as a means to direct the differentiation of stem cells for tissue engineering and regenerative medicine applications. In addition, these results suggest that the physical presence of microparticles within stem cell aggregates does not compromise PSC differentiation, but in fact the choice of biomaterials can impact the propensity of stem cells to adopt particular differentiated cell phenotypes. PMID:20864164

Protein aggregation and prionopathies.

PubMed

Renner, M; Melki, R

2014-06-01

Prion protein and prion-like proteins share a number of characteristics. From the molecular point of view, they are constitutive proteins that aggregate following conformational changes into insoluble particles. These particles escape the cellular clearance machinery and amplify by recruiting the soluble for of their constituting proteins. The resulting protein aggregates are responsible for a number of neurodegenerative diseases such as Creutzfeldt-Jacob, Alzheimer, Parkinson and Huntington diseases. In addition, there are increasing evidences supporting the inter-cellular trafficking of these aggregates, meaning that they are "transmissible" between cells. There are also evidences that brain homogenates from individuals developing Alzheimer and Parkinson diseases propagate the disease in recipient model animals in a manner similar to brain extracts of patients developing Creutzfeldt-Jacob's disease. Thus, the propagation of protein aggregates from cell to cell may be a generic phenomenon that contributes to the evolution of neurodegenerative diseases, which has important consequences on human health issues. Moreover, although the distribution of protein aggregates is characteristic for each disease, new evidences indicate the possibility of overlaps and crosstalk between the different disorders. Despite the increasing evidences that support prion or prion-like propagation of protein aggregates, there are many unanswered questions regarding the mechanisms of toxicity and this is a field of intensive research nowadays. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Quantification of the aggregation of magnetic nanoparticles with different polymeric coatings in cell culture medium

NASA Astrophysics Data System (ADS)

Eberbeck, D.; Kettering, M.; Bergemann, C.; Zirpel, P.; Hilger, I.; Trahms, L.

2010-10-01

The knowledge of the physico-chemical characteristics of magnetic nanoparticles (MNPs) is essential to enhance the efficacy of MNP-based therapeutic treatments (e.g. magnetic heating, magnetic drug targeting). According to the literature, the MNP uptake by cells may depend on the coating of MNPs, the surrounding medium as well as on the aggregation behaviour of the MNPs. Therefore, in this study, the aggregation behaviour of MNPs in various media was investigated. MNPs with different coatings were suspended in cell culture medium (CCM) containing fetal calf serum (FCS) and the distribution of the hydrodynamic sizes was measured by magnetorelaxometry (MRX). FCS as well as bovine serum albumin (BSA) buffer (phosphate buffered saline with 0.1% bovine serum albumin) may induce MNP aggregation. Its strength depends crucially on the type of coating. The degree of aggregation in CCM depends on its FCS content showing a clear, local maximum at FCS concentrations, where the IgG concentration (part of FCS) is of the order of the MNP number concentration. Thus, we attribute the observed aggregation behaviour to the mechanism of agglutination of MNPs by serum compartments as for example IgG. No aggregation was induced for MNPs coated with dextran, polyarabic acid or sodium phosphate, respectively, which were colloidally stable in CCM.

NFκB is a central regulator of protein quality control in response to protein aggregation stresses via autophagy modulation

PubMed Central

Nivon, Mathieu; Fort, Loïc; Muller, Pascale; Richet, Emma; Simon, Stéphanie; Guey, Baptiste; Fournier, Maëlenn; Arrigo, André-Patrick; Hetz, Claudio; Atkin, Julie D.; Kretz-Remy, Carole

2016-01-01

During cell life, proteins often misfold, depending on particular mutations or environmental changes, which may lead to protein aggregates that are toxic for the cell. Such protein aggregates are the root cause of numerous diseases called “protein conformational diseases,” such as myofibrillar myopathy and familial amyotrophic lateral sclerosis. To fight against aggregates, cells are equipped with protein quality control mechanisms. Here we report that NFκB transcription factor is activated by misincorporation of amino acid analogues into proteins, inhibition of proteasomal activity, expression of the R120G mutated form of HspB5 (associated with myofibrillar myopathy), or expression of the G985R and G93A mutated forms of superoxide dismutase 1 (linked to familial amyotrophic lateral sclerosis). This noncanonical stimulation of NFκB triggers the up-regulation of BAG3 and HspB8 expression, two activators of selective autophagy, which relocalize to protein aggregates. Then NFκB-dependent autophagy allows the clearance of protein aggregates. Thus NFκB appears as a central and major regulator of protein aggregate clearance by modulating autophagic activity. In this context, the pharmacological stimulation of this quality control pathway might represent a valuable strategy for therapies against protein conformational diseases. PMID:27075172

Protein aggregation in bacteria: the thin boundary between functionality and toxicity.

PubMed

Bednarska, Natalia G; Schymkowitz, Joost; Rousseau, Frederic; Van Eldere, Johan

2013-09-01

Misfolding and aggregation of proteins have a negative impact on all living organisms. In recent years, aggregation has been studied in detail due to its involvement in neurodegenerative diseases, including Alzheimer's, Parkinson's and Huntington's diseases, and type II diabetes--all associated with accumulation of amyloid fibrils. This research highlighted the central importance of protein homeostasis, or proteostasis for short, defined as the cellular state in which the proteome is both stable and functional. It implicates an equilibrium between synthesis, folding, trafficking, aggregation, disaggregation and degradation. In accordance with the eukaryotic systems, it has been documented that protein aggregation also reduces fitness of bacterial cells, but although our understanding of the cellular protein quality control systems is perhaps most detailed in bacteria, the use of bacterial proteostasis as a drug target remains little explored. Here we describe protein aggregation as a normal physiological process and its role in bacterial virulence and we shed light on how bacteria defend themselves against the toxic threat of aggregates. We review the impact of aggregates on bacterial viability and look at the ways that bacteria use to maintain a balance between aggregation and functionality. The proteostasis in bacteria can be interrupted via overexpression of proteins, certain antibiotics such as aminoglycosides, as well as antimicrobial peptides--all leading to loss of cell viability. Therefore intracellular protein aggregation and disruption of proteostatic balance in bacteria open up another strategy that should be explored towards the discovery of new antimicrobials.

Red blood cell aggregation changes are depended on its initial value: Effect of long-term drug treatment and short-term cell incubation with drug.

PubMed

Muravyov, A V; Tikhomirova, I A; Maimistova, A A; Bulaeva, S V; Mikhailov, P V; Kislov, N V

2011-01-01

This study was designed to investigate whether the red cell aggregation depends on its initial level under drug therapy or cell incubation with bioactive chemical compounds. Sixty six subjects were enrolled onto this study, and sub-divided into two groups: the first group of patients (n = 36) with cerebral atherosclerosis received pentoxifylline therapy (400 mg, thrice daily) for 4 weeks. The patients of the second group were initially treated with Epoetin beta 10,000 units subcutaneously thrice a week, for 4 weeks. The second group - adult anemic patients (n = 30) with the confirmed diagnosis of solid cancer (Hb < 100 g/L). After 4 weeks of pentoxifylline treatment the red cell aggregation increased (p < 0.05) in the patients with initially low RBCA. On the other hand in the patients with initially high RBCA treatment with pentoxifylline reduced it markedly (p < 0.01). In vitro experiments with pentoxifylline RBC incubation resulted in a decrease of the initially high RBCA by 47% (p < 0.01), whereas in the sub-group with initially low RBCA it increased. It was observed that after 4 weeks of epoetin-beta treatment 75% the anemic patients with initially high RBCA had an aggregation lowering. The drop of aggregation was about 34% (p < 0.01). At the same time 25% of the study patients had a significant RBCA increase (p < 0.05) after treatment. The initially low red cell aggregation after incubation with epoetin-beta was markedly increased by 122% (p < 0.05). On the contrary initially high RBCA was reduced by 47% (p < 0.05). When forskolin (10 μM) was added to the RBC suspensions the RBCA was increased in sub-group of subjects with initially low aggregation and it was decreased in sub-group with initially high one. The similar RBCA changes were observed when RBC suspensions were incubated with vinpocetine, calcium ionophore (A23187), Phorbol 12-myristate 13-acetate (PMA) as a protein kinase C (PKC) stimulator. A major finding of this study is that the red cell aggregation effects of some drugs depend markedly on the initial, pre-treatment aggregation status of the patients. These results demonstrate that the different red blood cell aggregation responses to the biological stimuli depend strongly on the initial, pre-treatment status of the subject and the most probably it is connected with the crosstalk between the adenylyl cyclase signaling pathway and Ca2+ regulatory mechanism.

Interaction and Aggregation of Colloidal Biological Particles and Droplets in Electrically-Driven Flows

NASA Technical Reports Server (NTRS)

Davis, Robert H.; Loewenberg, Michael

1997-01-01

The primary objective of this research was to develop a fundamental understanding of aggregation and coalescence processes during electrically-driven migration of cells, particles and droplets. The process by which charged cells, particles, molecules, or drops migrate in a weak electric field is known as electrophoresis. If the migrating species have different charges or surface potentials, they will migrate at different speeds and thus may collide and aggregate or coalesce. Aggregation and coalescence are undesirable, if the goal is to separate the different species on the basis of their different electrophoretic mobilities.

Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality.

PubMed

Buemo, Carla Paola; Gambini, Andrés; Moro, Lucia Natalia; Hiriart, María Inés; Fernández-Martín, Rafael; Collas, Philippe; Salamone, Daniel Felipe

2016-01-01

In this study, we analyzed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst diameter and cell number, DNA fragmentation levels and the expression of genes associated with pluripotency, apoptosis, trophoblast and DNA methylation in the porcine. Zona-free reconstructed cloned embryos were cultured in the well of the well system, placing one (1x non aggregated group) or three (3x group) embryos per microwell. Our results showed that aggregation of three embryos increased blastocyst formation rate and blastocyst diameter of cloned pig embryos. DNA fragmentation levels in 3x aggregated cloned blastocysts were significantly decreased compared to 1x blastocysts. Levels of Oct4, Klf4, Igf2, Bax and Dnmt 1 transcripts were significantly higher in aggregated embryos, whereas Nanog levels were not affected. Transcripts of Cdx2 and Bcl-xl were essentially non-detectable. Our study suggests that embryo aggregation in the porcine may be beneficial for cloned embryo development and embryo quality, through a reduction in apoptotic levels and an improvement in cell reprogramming.

State-of-the-Art Fluorescence Fluctuation-Based Spectroscopic Techniques for the Study of Protein Aggregation

PubMed Central

Kinjo, Masataka

2018-01-01

Neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease, are devastating proteinopathies with misfolded protein aggregates accumulating in neuronal cells. Inclusion bodies of protein aggregates are frequently observed in the neuronal cells of patients. Investigation of the underlying causes of neurodegeneration requires the establishment and selection of appropriate methodologies for detailed investigation of the state and conformation of protein aggregates. In the current review, we present an overview of the principles and application of several methodologies used for the elucidation of protein aggregation, specifically ones based on determination of fluctuations of fluorescence. The discussed methods include fluorescence correlation spectroscopy (FCS), imaging FCS, image correlation spectroscopy (ICS), photobleaching ICS (pbICS), number and brightness (N&B) analysis, super-resolution optical fluctuation imaging (SOFI), and transient state (TRAST) monitoring spectroscopy. Some of these methodologies are classical protein aggregation analyses, while others are not yet widely used. Collectively, the methods presented here should help the future development of research not only into protein aggregation but also neurodegenerative diseases. PMID:29570669

Embryo aggregation does not improve the development of interspecies somatic cell nuclear transfer embryos in the horse.

PubMed

Gambini, Andrés; De Stéfano, Adrián; Jarazo, Javier; Buemo, Carla; Karlanian, Florencia; Salamone, Daniel Felipe

2016-09-01

The low efficiency of interspecies somatic cell nuclear transfer (iSCNT) makes it necessary to investigate new strategies to improve embryonic developmental competence. Embryo aggregation has been successfully applied to improve cloning efficiency in mammals, but it remains unclear whether it could also be beneficial for iSCNT. In this study, we first compared the effect of embryo aggregation over in vitro development and blastocyst quality of porcine, bovine, and feline zona-free (ZF) parthenogenetic (PA) embryos to test the effects of embryo aggregation on species that were later used as enucleated oocytes donors in our iSCNT study. We then assessed whether embryo aggregation could improve the in vitro development of ZF equine iSCNT embryos after reconstruction with porcine, bovine, and feline ooplasm. Bovine- and porcine-aggregated PA blastocysts had significantly larger diameters compared with nonaggregated embryos. On the other hand, feline- and bovine-aggregated PA embryos had higher blastocyst cell number. Embryo aggregation of equine-equine SCNT was found to be beneficial for embryo development as we have previously reported, but the aggregation of three ZF reconstructed embryos did not improve embryo developmental rates on iSCNT. In vitro embryo development of nonaggregated iSCNT was predominantly arrested around the stage when transcriptional activation of the embryonic genome is reported to start on the embryo of the donor species. Nevertheless, independent of embryo aggregation, equine blastocyst-like structures could be obtained in our study using domestic feline-enucleated oocytes. Taken together, these results reported that embryo aggregation enhance in vitro PA embryo development and embryo quality but effects vary depending on the species. Embryo aggregation also improves, as expected, the in vitro embryo development of equine-equine SCNT embryos; however, we did not observe positive effects on equine iSCNT embryo development. Among oocytes from domestic animals tested in our study, the feline ooplasm might be the most appropriate recipient to partially allow preimplantation embryo development of iSCNT equine embryos. Copyright © 2016 Elsevier Inc. All rights reserved.

A plant cell-based system that predicts aβ42 misfolding: potential as a drug discovery tool for Alzheimer's disease.

PubMed

Zhao, Tiehan; Zeng, Ying; Kermode, Allison R

2012-11-01

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the accumulation of amyloid β (Aβ) peptides and the failure of mechanisms to clear toxic aggregates. The Aβ42 peptide is considered to be a causative factor that underlies the pathophysiology of AD, in part due to its propensity for misfolding and aggregation; the small oligomers that result represent toxic species. Thus agents that prevent Aβ42 misfolding/aggregation or, alternatively improve Aβ42 oligomer clearance, may have significant therapeutic value. We have developed the basis for a drug screening system based on transgenic plant cells that express Aβ42 fusion proteins to serve as the reliable indicators of the general conformational status of Aβ42. Within cells of transgenic tobacco and Nicotiana benthamiana, misfolding of Aβ42 causes the misfolding of a GFP fusion partner, and consequently there is a loss of fluorescence associated with the native GFP protein. In a similar fusion consisting of Aβ42 linked to hygromycin phosphotransferase II (Hpt II), a hygromycin-resistance marker, misfolding of Aβ42 leads to a misfolded Hpt II, and consequently the transgenic cells are unable to grow on media containing hygromycin. Importantly, substitution of the 'aggregation-prone' Aβ42 with a missense mutant of Aβ42 (F19S/L34F) that is not prone to misfolding/aggregation, 'rescues' both fusion partners. Several 'positive control' chemicals that represent inhibitors of Aβ42 aggregation, including curcumin, epigallocatechin-3-gallate (EGCG), and resveratrol show efficacy in preventing the Aβ42-fusion proteins from misfolding/aggregating in the transgenic plant cells. We discuss the potential of the two fusion protein systems to serve as the basis for an inexpensive, selective, and efficient screening system in which a plant cell can fluoresce or survive only in the presence of drug candidates that are able to prevent Aβ42 misfolding/aggregation. Copyright © 2012 Elsevier Inc. All rights reserved.

Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro.

PubMed

Baillie-Johnson, Peter; van den Brink, Susanne Carina; Balayo, Tina; Turner, David Andrew; Martinez Arias, Alfonso

2015-11-24

We have developed a protocol improving current Embryoid Body (EB) culture which allows the study of self-organization, symmetry breaking, axial elongation and cell fate specification using aggregates of mouse embryonic stem cells (mESCs) in suspension culture. Small numbers of mESCs are aggregated in basal medium for 48 hr in non-tissue-culture-treated, U-bottomed 96-well plates, after which they are competent to respond to experimental signals. Following treatment, these aggregates begin to show signs of polarized gene expression and gradually alter their morphology from a spherical mass of cells to an elongated, well organized structure in the absence of external asymmetry cues. These structures are not only able to display markers of the three germ layers, but actively display gastrulation-like movements, evidenced by a directional dislodgement of individual cells from the aggregate, which crucially occurs at one region of the elongated structure. This protocol provides a detailed method for the reproducible formation of these aggregates, their stimulation with signals such as Wnt/β-Catenin activation and BMP inhibition and their analysis by single time-point or time-lapse fluorescent microscopy. In addition, we describe modifications to current whole-mount mouse embryo staining procedures for immunocytochemical analysis of specific markers within fixed aggregates. The changes in morphology, gene expression and length of the aggregates can be quantitatively measured, providing information on how signals can alter axial fates. It is envisaged that this system can be applied both to the study of early developmental events such as axial development and organization, and more broadly, the processes of self-organization and cellular decision-making. It may also provide a suitable niche for the generation of cell types present in the embryo that are unobtainable from conventional adherent culture such as spinal cord and motor neurones.

How Do the Size, Charge and Shape of Nanoparticles Affect Amyloid β Aggregation on Brain Lipid Bilayer?

NASA Astrophysics Data System (ADS)

Kim, Yuna; Park, Ji-Hyun; Lee, Hyojin; Nam, Jwa-Min

2016-01-01

Here, we studied the effect of the size, shape, and surface charge of Au nanoparticles (AuNPs) on amyloid beta (Aβ) aggregation on a total brain lipid-based supported lipid bilayer (brain SLB), a fluid platform that facilitates Aβ-AuNP aggregation process. We found that larger AuNPs induce large and amorphous aggregates on the brain SLB, whereas smaller AuNPs induce protofibrillar Aβ structures. Positively charged AuNPs were more strongly attracted to Aβ than negatively charged AuNPs, and the stronger interactions between AuNPs and Aβ resulted in fewer β-sheets and more random coil structures. We also compared spherical AuNPs, gold nanorods (AuNRs), and gold nanocubes (AuNCs) to study the effect of nanoparticle shape on Aβ aggregation on the brain SLB. Aβ was preferentially bound to the long axis of AuNRs and fewer fibrils were formed whereas all the facets of AuNCs interacted with Aβ to produce the fibril networks. Finally, it was revealed that different nanostructures induce different cytotoxicity on neuroblastoma cells, and, overall, smaller Aβ aggregates induce higher cytotoxicity. The results offer insight into the roles of NPs and brain SLB in Aβ aggregation on the cell membrane and can facilitate the understanding of Aβ-nanostructure co-aggregation mechanism and tuning Aβ aggregate structures.

Determinants of platelet aggregation in 50-70-year-old men from three Japanese communities.

PubMed

Imano, Hironori; Iso, Hiroyasu; Sato, Shinichi; Kitamura, Akihiko; Okamura, Tomonori; Tanigawa, Takeshi; Ohira, Tetsuya; Kudo, Minako; Naito, Yoshihiko; Iida, Minoru; Shimamoto, Takashi

2002-12-01

To investigate the association of lifestyle and constitutional variables with platelet aggregation, we examined the platelet aggregation, serum fatty acid composition, alcohol intake, smoking, and dietary intake of seafood and soybean estimated by a 1-week dietary record in 448 males aged 50-70 in three rural Japanese communities: Ikawa, Akita prefecture (northeast coast), Noichi, Kochi prefecture (southwest coast), and Kyowa, Ibaraki prefecture (central inland). Platelet aggregatory threshold index (PATI) was used to determine the minimum concentration of adenosine 5'-diphosphate (ADP) that caused a non-reversible aggregation of platelets. Intake of seafood and n3-polyunsaturated fatty acid and ingestion of ethanol were higher in the northeast coastal community than in the other two communities. Mean platelet and white blood cell counts were lower in northeast coastal community than in the other two communities. The geometric mean PATI was higher (i.e. platelet aggregation was lower) in the northeast coastal community than the other two communities. Within the entire sample, platelet aggregation correlated inversely with serum level of n3-polyunsaturated fatty acids and gamma-glutamyl transpeptidase, an index of alcohol consumption, and positively with platelet and white blood cell counts. Platelet aggregation tended to correlate positively with serum arachidonic acid. There was no correlation between smoking and platelet aggregation. Our results suggest that seafood intake and moderate alcohol consumption reduce platelet aggregation.

Active matter model of Myxococcus xanthus aggregation

NASA Astrophysics Data System (ADS)

Patch, Adam; Bahar, Fatmagul; Liu, Guannan; Thutupalli, Shashi; Welch, Roy; Yllanes, David; Shaevitz, Joshua; Marchetti, M. Cristina

Myxococcus xanthus is a soil-dwelling bacterium that exhibits several fascinating collective behaviors including streaming, swarming, and generation of fruiting bodies. A striking feature of M. xanthus is that it periodically reverses its motility direction. The first stage of fruiting body formation is characterized by the aggregation of cells on a surface into round mesoscopic structures. Experiments have shown that this aggregation relies heavily on regulation of the reversal rate and local mechanical interactions, suggesting motility-induced phase separation may play an important role. We have adapted self-propelled particle models to include cell reversal and motility suppression resulting from sporulation observed in aggregates. Using 2D molecular dynamics simulations, we map the phase behavior in the space of Péclet number and local density and examine the kinetics of aggregation for comparison to experiments.

Detection of pH-induced aggregation of "smart" gold nanoparticles with photothermal optical coherence tomography.

PubMed

Xiao, Peng; Li, Qingyun; Joo, Yongjoon; Nam, Jutaek; Hwang, Sekyu; Song, Jaejung; Kim, Sungjee; Joo, Chulmin; Kim, Ki Hean

2013-11-01

We report the feasibility of a novel contrast agent, namely "smart" gold nanoparticles (AuNPs), in the detection of cancer cells with photothermal optical coherence tomography (PT-OCT). "Smart" AuNPs form aggregation in low pH condition, which is typical for cancer cells, and this aggregation results in a shift of their absorption spectrum. A PT-OCT system was developed to detect this pH-induced aggregation by combining an OCT light source and a laser with 660 nm in wavelength for photothermal excitation. Optical detection of pH-induced aggregation was tested with solution samples at two different pH conditions. An increase in optical path length (OPL) variation was measured at mild acidic condition, while there was not much change at neutral condition. Detection of cancer cells was tested with cultured cell samples. HeLa and fibroblast cells, as cancer and normal cells respectively, were incubated with "smart" gold nanoparticles and measured with PT-OCT. An elevated OPL variation signal was detected with the HeLa cells while not much of a signal was detected with the fibroblast cells. With the novel optical property of "smart" AuNPs and high sensitivity of PT-OCT, this technique is promising for cancer cell detection.

Red blood cell deformability and aggregation behaviour in different animal species.

PubMed

Plasenzotti, R; Stoiber, B; Posch, M; Windberger, U

2004-01-01

Comparative animal studies showed the wide variation of whole blood and plasma viscosity, and erythrocyte aggregation among mammalian species. Whole blood viscosity and red blood cell aggregation is influenced by red cell fluidity. To evaluate differences in erythrocyte deformability in mammals, three species were investigated, whose erythrocytes have a different aggregation property: horse, as a species with high, dog with medium, and sheep with almost unmeasurable aggregation tendency. Erythrocyte deformability was tested ektacytometrically (Elongation Index [EI], LORCA, Mechatronics, Hoorn, Netherlands) at shear stresses from 0.30 to 53.06 Pa. Equine erythrocytes showed EI-values from 0.047 at low shear stress to 0.541 at high shear stress. The EI from dog's erythrocytes ranged from 0.035 to 0.595. Sheep's erythrocytes had an EI of 0.005 at low and 0.400 at high shear stress. Although it might be presumed from the aggregation property that horse had the highest EI among the three species, the EI of canine erythrocytes exceeded the value in horses by 10% at high shear stress. Further, equine erythrocytes started to deform at higher shear stresses (1.69 Pa) than did canine and ovine cells, whose EI increased continuously with increasing shear stress. At moderate shear stress (1-5 Pa) deformability was even higher in the sheep than in the horse. However, at shear stresses higher than 5.34 Pa, equine red cell elongation clearly exceeded the values of sheep. We conclude that erythrocyte elongation is different between the animal species, not clearly linked with the aggregation property, and that the degree of deformability at various shear stresses is species-specific.

Low-molecular weight heparin protamine complex augmented the potential of adipose-derived stromal cells to ameliorate limb ischemia.

PubMed

Kishimoto, Satoko; Inoue, Ken-Ichi; Nakamura, Shingo; Hattori, Hidemi; Ishihara, Masayuki; Sakuma, Masashi; Toyoda, Shigeru; Iwaguro, Hideki; Taguchi, Isao; Inoue, Teruo; Yoshida, Ken-Ichiro

2016-06-01

Heparin/protamine micro/nanoparticles (LH/P-MPs) were recently developed as low-molecular weight, biodegradable carriers for adipose-derived stromal cells (ADSCs). These particles can be used for a locally delivered stem cell therapy that promotes angiogenesis. LH/P-MPs bind to the cell surface of ADSCs and promote cell-to-cell interaction and aggregation of ADSCs. Cultured ADSC/LH/P-MP aggregates remain viable. Here, we examined the ability of these aggregates to rescue limb loss in a mouse model of hindlimb ischemia. Unilateral hindlimb ischemia was induced in adult male BALB/c mice by ligation of the iliac artery and hindlimb vein. For allotransplantation of ADSCs from the same inbred strain, we injected ADSC alone or ADSC/LH/P-MP aggregates or control medium (sham-treated) directly into the ischemic muscles. Ischemic limb blood perfusion, vessel density, and vessel area were recorded. The extent of ischemic limb necrosis or limb loss was assessed on postoperative days 2, 7, and 14. Compared with the sham-treatment control, treatment with ADSCs alone showed modest effects on blood perfusion recovery and increased the number of α-SMA-positive vessels. Response to ADSC/LH/P-MP aggregates was significantly greater than ADSCs alone for every endpoint. ADSC/LH/P-MP aggregates more effectively prevented the loss of ischemic hindlimbs than ADSCs alone or the sham-treatment. The LH/P-MPs augmented the effects of ADSCs on angiogenesis and reversal of limb ischemia. Use of ADSC/LH/P-MP aggregates offers a novel and convenient treatment method and potentially represents a promising new therapeutic approach to inducing angiogenesis in ischemic diseases. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

A prolyl oligopeptidase inhibitor, KYP-2047, reduces α-synuclein protein levels and aggregates in cellular and animal models of Parkinson's disease

PubMed Central

Myöhänen, TT; Hannula, MJ; Van Elzen, R; Gerard, M; Van Der Veken, P; García-Horsman, JA; Baekelandt, V; Männistö, PT; Lambeir, AM

2012-01-01

BACKGROUND AND PURPOSE The aggregation of α-synuclein is connected to the pathology of Parkinson's disease and prolyl oligopeptidase (PREP) accelerates the aggregation of α-synuclein in vitro. The aim of this study was to investigate the effects of a PREP inhibitor, KYP-2047, on α-synuclein aggregation in cell lines overexpressing wild-type or A30P/A53T mutant human α-syn and in the brains of two A30P α-synuclein transgenic mouse strains. EXPERIMENTAL APPROACH Cells were exposed to oxidative stress and then incubated with the PREP inhibitor during or after the stress. Wild-type or transgenic mice were treated for 5 days with KYP-2047 (2 × 3 mg·kg−1 a day). Besides immunohistochemistry and thioflavin S staining, soluble and insoluble α-synuclein protein levels were measured by Western blot. α-synuclein mRNA levels were quantified by PCR. The colocalization of PREP and α-synuclein,and the effect of KYP-2047 on cell viability were also investigated. KEY RESULTS In cell lines, oxidative stress induced a robust aggregation of α-synuclein,and low concentrations of KYP-2047 significantly reduced the number of cells with α-synuclein inclusions while abolishing the colocalization of α-synuclein and PREP. KYP-2047 significantly reduced the amount of aggregated α-synuclein,and it had beneficial effects on cell viability. In the transgenic mice, a 5-day treatment with the PREP inhibitor reduced the amount of α-synuclein immunoreactivity and soluble α-synuclein protein in the brain. CONCLUSIONS AND IMPLICATIONS The results suggest that the PREP may play a role in brain accumulation and aggregation of α-synuclein, while KYP-2047 seems to effectively prevent these processes. PMID:22233220

Functional and structural effects of amyloid-β aggregate on Xenopus laevis oocytes.

PubMed

Parodi, Jorge; Ochoa-de la Paz, Lenin; Miledi, Ricardo; Martínez-Torres, Ataúlfo

2012-10-01

Xenopus laevis oocytes exposed to amyloid-β aggregate generated oscillatory electric activity (blips) that was recorded by two-microelectrode voltage-clamp. The cells exhibited a series of "spontaneous" blips ranging in amplitude from 3.8 ± 0.9 nA at the beginning of the recordings to 6.8 ± 1.7 nA after 15 min of exposure to 1 μM aggregate. These blips were similar in amplitude to those induced by the channel-forming antimicrobial agents amphotericin B (7.8 ± 1.2 nA) and gramicidin (6.3 ± 1.1 nA). The amyloid aggregate-induced currents were abolished when extracellular Ca(2+) was removed from the bathing solution, suggesting a central role for this cation in generating the spontaneous electric activity. The amyloid aggregate also affected the Ca(2+)-dependent Cl(-) currents of oocytes, as shown by increased amplitude of the transient-outward chloride current (T(out)) and the serum-activated, oscillatory Cl(-) currents. Electron microcopy revealed that amyloid aggregate induced the dissociation of the follicular cells that surround the oocyte, thus leading to a failure in the electro-chemical communication between these cells. This was also evidenced by the suppression of the oscillatory Ca(2+)-dependent ATP-currents, which require proper coupling between oocytes and the follicular cell layer. These observations, made using the X. laevis oocytes as a versatile experimental model, may help to understand the effects of amyloid aggregate on cellular communication.

Elucidation of flow-mediated tumour cell-induced platelet aggregation using an ultrasound standing wave trap

PubMed Central

Bazou, D; Santos-Martinez, MJ; Medina, C; Radomski, MW

2011-01-01

BACKGROUND AND PURPOSE Tumour cells activate and aggregate platelets [tumour cell-induced platelet aggregation (TCIPA)] and this process plays an important role in the successful metastasis of cancer cells. To date, most studies on TCIPA have been conducted under no-flow conditions. In this study, we have investigated TCIPA in real time under flow conditions, using an ultrasound standing wave trap that allows formation and levitation of cancer cell clusters in suspension, thus mimicking the conditions generated by flowing blood. EXPERIMENTAL APPROACH Using 59M adenocarcinoma and HT1080 fibrosarcoma cells and human platelets, cancer cell cluster–platelet aggregates were imaged in real time using epi-fluorescence microscopy (F-actin) and investigated in detail using confocal microscopy (matrix metalloproteinase-2-GPIIb/IIIa co-localization) and scanning electron and helium-ion microscopy (<1 nm resolution). The release of gelatinases from aggregates was studied using zymography. KEY RESULTS We found that platelet activation and aggregation takes place on the surface of cancer cells (TCIPA), leading to time-dependent disruption of cancer cell clusters. Pharmacological modulation of TCIPA revealed that EDTA, prostacyclin, o-phenanthroline and apyrase significantly down-regulated TCIPA and, in turn, delayed cell cluster disruption, However, EGTA and aspirin were ineffective. Pharmacological inhibition of TCIPA correlated with the down-regulation of platelet activation as shown by flow-cytometry assay of platelet P-selectin. CONCLUSION AND IMPLICATIONS Our results show for the first time, that during TCIPA, platelet activation disrupts cancer cell clusters and this can contribute to metastasis. Thus, selective targeting of platelet aggregate–cancer cell clusters may be an important strategy to control metastasis. PMID:21182493

Cationic Surface Charge Combined with Either Vitronectin or Laminin Dictates the Evolution of Human Embryonic Stem Cells/Microcarrier Aggregates and Cell Growth in Agitated Cultures

PubMed Central

Lam, Alan Tin-Lun; Li, Jian; Chen, Allen Kuan-Liang; Reuveny, Shaul

2014-01-01

The expansion of human pluripotent stem cells (hPSC) for biomedical applications generally compels a defined, reliable, and scalable platform. Bioreactors offer a three-dimensional culture environment that relies on the implementation of microcarriers (MC), as supports for cell anchorage and their subsequent growth. Polystyrene microspheres/MC coated with adhesion-promoting extracellular matrix (ECM) protein, vitronectin (VN), or laminin (LN) have been shown to support hPSC expansion in a static environment. However, they are insufficient to promote human embryonic stem cells (hESC) seeding and their expansion in an agitated environment. The present study describes an innovative technology, consisting of a cationic charge that underlies the ECM coatings. By combining poly-L-lysine (PLL) with a coating of ECM protein, cell attachment efficiency and cell spreading are improved, thus enabling seeding under agitation in a serum-free medium. This coating combination also critically enables the subsequent formation and evolution of hPSC/MC aggregates, which ensure cell viability and generate high yields. Aggregate dimensions of at least 300 μm during early cell growth give rise to ≈15-fold expansion at 7 days' culture. Increasing aggregate numbers at a quasi-constant size of ≈300 μm indicates hESC growth within a self-regulating microenvironment. PLL+LN enables cell seeding and aggregate evolution under constant agitation, whereas PLL+VN requires an intermediate 2-day static pause to attain comparable aggregate sizes and correspondingly high expansion yields. The cells' highly reproducible bioresponse to these defined and characterized MC surface properties is universal across multiple cell lines, thus confirming the robustness of this scalable expansion process in a defined environment. PMID:24641164

Effect of cobalt ions on the interaction between macrophages and titanium.

PubMed

Pettersson, Mattias; Pettersson, Jean; Thorén, Margareta Molin; Johansson, Anders

2018-04-30

Inflammation and bone reduction around dental implants are described as peri-implantitis and can be caused by an inflammatory response against bacterial products and toxins. Titanium (Ti) forms aggregates with serum proteins, which activate and cause release of the cytokine interleukin (IL-1β) from human macrophages. It was hypothesized that cobalt (Co) ions can interact in the formation of pro-inflammatory aggregates, formed by titanium. To test this hypothesis, we differentiated THP-1 cells into macrophages and exposed them to Ti ions alone or in combination with Co ions to investigate if IL-1β release and cytotoxicity were affected. We also investigated aggregate formation, cell uptake and human biopsies with inductively coupled plasma atomic emission spectroscopy (ICP-AES) and electron microscopy. Co at a concentration of 100 µM neutralized the IL-1β release from human macrophages and affected the aggregate formation. The aggregates formed by Ti could be detected in the cytosol of macrophages. In the presence of Co, the Ti-induced aggregates were located in the cytosol of the cultured macrophages, but outside the lysosomal structures. It is concluded that Co can neutralize the Ti-induced activation and release of active IL-1β from human macrophages in vitro. Also, serum proteins are needed for the formation of metal-protein aggregates in cell medium. Furthermore, the structures of the aggregates as well as the localisation after cellular uptake differ if Co is present in a Ti solution. Phagocytized aggregates with a similar appearance seen in vitro with Ti present, were also visible in a sample from human peri-implant tissue. This article is protected by copyright. All rights reserved. © 2018 Wiley Periodicals, Inc.

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

Size analysis of polyglutamine protein aggregates using fluorescence detection in an analytical ultracentrifuge.

PubMed

Polling, Saskia; Hatters, Danny M; Mok, Yee-Foong

2013-01-01

Defining the aggregation process of proteins formed by poly-amino acid repeats in cells remains a challenging task due to a lack of robust techniques for their isolation and quantitation. Sedimentation velocity methodology using fluorescence detected analytical ultracentrifugation is one approach that can offer significant insight into aggregation formation and kinetics. While this technique has traditionally been used with purified proteins, it is now possible for substantial information to be collected with studies using cell lysates expressing a GFP-tagged protein of interest. In this chapter, we describe protocols for sample preparation and setting up the fluorescence detection system in an analytical ultracentrifuge to perform sedimentation velocity experiments on cell lysates containing aggregates formed by poly-amino acid repeat proteins.

Chemically mediated group formation in soil-dwelling larvae and pupae of the beetle Trypoxylus dichotomus

NASA Astrophysics Data System (ADS)

Kojima, Wataru; Ishikawa, Yukio; Takanashi, Takuma

2014-09-01

Many insects form groups through interactions among individuals, and these are often mediated by chemical, acoustic, or visual cues and signals. In spite of the diversity of soil-dwelling insects, their aggregation behaviour has not been examined as extensively as that of aboveground species. We investigated the aggregation mechanisms of larvae of the Japanese rhinoceros beetle Trypoxylus dichotomus, which live in groups in humus soil. In two-choice laboratory tests, 2nd- and 3rd-instar larvae gathered at conspecific larvae irrespective of the kinship. The ablation of maxillae, which bear chemosensilla, abolished aggregation behaviour. Intact larvae also exhibited aggregation behaviour towards a larval homogenate. These results suggest that larval aggregation is mediated by chemical cues. We also demonstrated that the mature larvae of T. dichotomus built their pupal cells close to a mesh bag containing a conspecific pupal cell, which indicated that larvae utilize chemical cues emanating from these cells to select the pupation site. Thus, the larvae of T. dichotomus may use chemical cues from the conspecifics in two different contexts, i.e. larval aggregation and pupation site selection. Using conspecific cues, larvae may be able to choose suitable locations for foraging or building pupal cells. The results of the present study highlight the importance of chemical information in belowground ecology.

Chemically mediated group formation in soil-dwelling larvae and pupae of the beetle Trypoxylus dichotomus.

PubMed

Kojima, Wataru; Ishikawa, Yukio; Takanashi, Takuma

2014-09-01

Many insects form groups through interactions among individuals, and these are often mediated by chemical, acoustic, or visual cues and signals. In spite of the diversity of soil-dwelling insects, their aggregation behaviour has not been examined as extensively as that of aboveground species. We investigated the aggregation mechanisms of larvae of the Japanese rhinoceros beetle Trypoxylus dichotomus, which live in groups in humus soil. In two-choice laboratory tests, 2nd- and 3rd-instar larvae gathered at conspecific larvae irrespective of the kinship. The ablation of maxillae, which bear chemosensilla, abolished aggregation behaviour. Intact larvae also exhibited aggregation behaviour towards a larval homogenate. These results suggest that larval aggregation is mediated by chemical cues. We also demonstrated that the mature larvae of T. dichotomus built their pupal cells close to a mesh bag containing a conspecific pupal cell, which indicated that larvae utilize chemical cues emanating from these cells to select the pupation site. Thus, the larvae of T. dichotomus may use chemical cues from the conspecifics in two different contexts, i.e. larval aggregation and pupation site selection. Using conspecific cues, larvae may be able to choose suitable locations for foraging or building pupal cells. The results of the present study highlight the importance of chemical information in belowground ecology.

Staurosporine induces rapid homotypic intercellular adhesion of U937 cells via multiple kinase activation

PubMed Central

Cho, Jae Youl; Katz, David R; Chain, Benjamin M

2003-01-01

Staurosporine is a broad-specificity kinase inhibitor, which has acted as lead compound for the development of some novel cytotoxic compounds for treatment of cancer. This study investigates the unexpected observation that staurosporine can also induce homotypic cellular aggregation. In this study, staurosporine is shown to activate rapid homotypic aggregation of U937 cells, at concentrations below those required to induce cell death. This activity is a particular feature of staurosporine, and is not shared by a number of other kinase inhibitors. The proaggregating activity of staurosporine is inhibited by deoxyglucose, cytochalasin B and colchicine. Staurosporine-induced aggregation can be distinguished from that induced by the phorbol 12-myristate 13-acetate by faster kinetics and insensitivity to cycloheximide. Staurosporine induces translocation of conventional and novel, but not atypical isoforms of protein kinase C (PKC). Aggregation induced by staurosporine is inhibited by a number of inhibitors of PKC isoforms, and by inhibitors of protein tyrosine kinases. Staurosporine also induces rapid phosphorylation of ERK and p38, and inhibitors of both these enzymes block aggregation. Staurosporine induces dysregulated activation of multiple kinase signaling pathways in U937 cells, and the combined activity of several of these pathways is essential for the induction of aggregation. PMID:12970105

Tracking calcification in tissue-engineered bone using synchrotron micro-FTIR and SEM.

PubMed

Deegan, Anthony J; Cinque, Gianfelice; Wehbe, Katia; Konduru, Sandeep; Yang, Ying

2015-02-01

One novel tissue engineering approach to mimic in vivo bone formation is the use of aggregate or micromass cultures. Various qualitative and quantitative techniques, such as histochemical staining, protein assay kits and RT-PCR, have been used previously on cellular aggregate studies to investigate how these intricate arrangements lead to mature bone tissue. However, these techniques struggle to reveal spatial and temporal distribution of proliferation and mineralization simultaneously. Synchrotron-based Fourier transform infrared microspectroscopy (micro-FTIR) offers a unique insight at the molecular scale by coupling high IR sensitivity to organic matter with the high spatial resolution allowed by diffraction limited SR microbeam. This study is set to investigate the effects of culture duration and aggregate size on the dynamics and spatial distribution of calcification in engineered bone aggregates by a combination of micro-FTIR and scanning electron microscopy (SEM)/energy-dispersive X-ray spectroscopy (EDX). A murine bone cell line has been used, and small/large bone aggregates have been induced using different chemically treated culture substrates. Our findings suggest that bone cell aggregate culturing can greatly increase levels of mineralization over short culture periods. The size of the aggregates influences mineralisation rates with larger aggregates mineralizing at a faster rate than their smaller counterparts. The micro-FTIR mapping has demonstrated that mineralization in the larger aggregates initiated from the periphery and spread to the centre, whilst the smaller aggregates have more minerals in the centre at the early stage and deposited more in the periphery after further culturing, implying that aggregate size influences calcification distribution and development over time. SEM/EDX data correlates well with the micro-FTIR results for the total mineral content. Thus, synchrotron-based micro-FTIR can accurately track mineralization process/mechanism in the engineered bone.

RCCS bioreactor-based modelled microgravity induces significant changes on in vitro 3D neuroglial cell cultures.

PubMed

Morabito, Caterina; Steimberg, Nathalie; Mazzoleni, Giovanna; Guarnieri, Simone; Fanò-Illic, Giorgio; Mariggiò, Maria A

2015-01-01

We propose a human-derived neuro-/glial cell three-dimensional in vitro model to investigate the effects of microgravity on cell-cell interactions. A rotary cell-culture system (RCCS) bioreactor was used to generate a modelled microgravity environment, and morphofunctional features of glial-like GL15 and neuronal-like SH-SY5Y cells in three-dimensional individual cultures (monotypic aggregates) and cocultures (heterotypic aggregates) were analysed. Cell survival was maintained within all cell aggregates over 2 weeks of culture. Moreover, compared to cells as traditional static monolayers, cell aggregates cultured under modelled microgravity showed increased expression of specific differentiation markers (e.g., GL15 cells: GFAP, S100B; SH-SY5Y cells: GAP43) and modulation of functional cell-cell interactions (e.g., N-CAM and Cx43 expression and localisation). In conclusion, this culture model opens a wide range of specific investigations at the molecular, biochemical, and morphological levels, and it represents an important tool for in vitro studies into dynamic interactions and responses of nervous system cell components to microgravity environmental conditions.

RCCS Bioreactor-Based Modelled Microgravity Induces Significant Changes on In Vitro 3D Neuroglial Cell Cultures

PubMed Central

Mazzoleni, Giovanna; Fanò-Illic, Giorgio; Mariggiò, Maria A.

2015-01-01

We propose a human-derived neuro-/glial cell three-dimensional in vitro model to investigate the effects of microgravity on cell-cell interactions. A rotary cell-culture system (RCCS) bioreactor was used to generate a modelled microgravity environment, and morphofunctional features of glial-like GL15 and neuronal-like SH-SY5Y cells in three-dimensional individual cultures (monotypic aggregates) and cocultures (heterotypic aggregates) were analysed. Cell survival was maintained within all cell aggregates over 2 weeks of culture. Moreover, compared to cells as traditional static monolayers, cell aggregates cultured under modelled microgravity showed increased expression of specific differentiation markers (e.g., GL15 cells: GFAP, S100B; SH-SY5Y cells: GAP43) and modulation of functional cell-cell interactions (e.g., N-CAM and Cx43 expression and localisation). In conclusion, this culture model opens a wide range of specific investigations at the molecular, biochemical, and morphological levels, and it represents an important tool for in vitro studies into dynamic interactions and responses of nervous system cell components to microgravity environmental conditions. PMID:25654124

Cytosolic Proteostasis via Importing of Misfolded Proteins into Mitochondria

PubMed Central

Ruan, Linhao; Zhou, Chuankai; Jin, Erli; Kucharavy, Andrei; Zhang, Ying; Wen, Zhihui; Florens, Laurence; Li, Rong

2017-01-01

Loss of proteostasis underlies aging and neurodegeneration characterized by the accumulation of protein aggregates and mitochondrial dysfunction1–5. Although many neurodegenerative-disease proteins can be found in mitochondria4,6, it remains unclear how these disease manifestations may be related. In yeast, protein aggregates formed under stress or during aging are preferentially retained by the mother cell in part through tethering to mitochondria, while the disaggregase Hsp104 helps dissociate aggregates to enable refolding or degradation of misfolded proteins7–10. Here we show that in yeast cytosolic proteins prone to aggregation are imported into mitochondria for degradation. Protein aggregates formed under heat shock (HS) contain both cytosolic and mitochondrial proteins and interact with mitochondrial import complex. Many aggregation-prone proteins enter mitochondrial intermembrane space and matrix after HS, while some do so even without stress. Timely dissolution of cytosolic aggregates requires mitochondrial import machinery and proteases. Blocking mitochondrial import but not the proteasome activity causes a marked delay in the degradation of aggregated proteins. Defects in cytosolic Hsp70s leads to enhanced entry of misfolded proteins into mitochondria and elevated mitochondrial stress. We term this mitochondria-mediated proteostasis mechanism MAGIC (mitochondria as guardian in cytosol) and provide evidence that it may exist in human cells. PMID:28241148

Focal high cell density generates a gradient of patterns in self-organizing vascular mesenchymal cells.

PubMed

Cheng, Henry; Reddy, Aneela; Sage, Andrew; Lu, Jinxiu; Garfinkel, Alan; Tintut, Yin; Demer, Linda L

2012-01-01

In embryogenesis, structural patterns, such as vascular branching, may form via a reaction-diffusion mechanism in which activator and inhibitor morphogens guide cells into periodic aggregates. We previously found that vascular mesenchymal cells (VMCs) spontaneously aggregate into nodular structures and that morphogen pairs regulate the aggregation into patterns of spots and stripes. To test the effect of a focal change in activator morphogen on VMC pattern formation, we created a focal zone of high cell density by plating a second VMC layer within a cloning ring over a confluent monolayer. After 24 h, the ring was removed and pattern formation monitored by phase-contrast microscopy. At days 2-8, the patterns progressed from uniform distributions to swirl, labyrinthine and spot patterns. Within the focal high-density zone (HDZ) and a narrow halo zone, cells aggregated into spot patterns, whilst in the outermost zone of the plate, cells formed a labyrinthine pattern. The area occupied by aggregates was significantly greater in the outermost zone than in the HDZ or halo. The rate of pattern progression within the HDZ increased as a function of its plating density. Thus, focal differences in cell density may drive pattern formation gradients in tissue architecture, such as vascular branching. Copyright © 2012 S. Karger AG, Basel.

Anti-aging treatments slow propagation of synucleinopathy by restoring lysosomal function.

PubMed

Kim, Dong-Kyu; Lim, Hee-Sun; Kawasaki, Ichiro; Shim, Yhong-Hee; Vaikath, Nishant N; El-Agnaf, Omar M A; Lee, He-Jin; Lee, Seung-Jae

2016-10-02

Aging is the major risk factor for neurodegenerative diseases that are also associated with impaired proteostasis, resulting in abnormal accumulation of protein aggregates. However, the role of aging in development and progression of disease remains elusive. Here, we used Caenorhabditis elegans models to show that aging-promoting genetic variations accelerated the rate of cell-to-cell transmission of SNCA/α-synuclein aggregates, hallmarks of Parkinson disease, and the progression of disease phenotypes, such as nerve degeneration, behavioral deficits, and reduced life span. Genetic and pharmacological anti-aging manipulations slowed the spread of aggregates and the associated phenotypes. Lysosomal degradation was significantly impaired in aging models, while anti-aging treatments reduced the impairment. Transgenic expression of hlh-30p::hlh-30, the master controller of lysosomal biogenesis, alleviated intercellular transmission of aggregates in the aging model. Our results demonstrate that the rate of aging closely correlates with the rate of aggregate propagation and that general anti-aging treatments can slow aggregate propagation and associated disease progression by restoring lysosomal function.

Real-time imaging of Huntingtin aggregates diverting target search and gene transcription

PubMed Central

Li, Li; Liu, Hui; Dong, Peng; Li, Dong; Legant, Wesley R; Grimm, Jonathan B; Lavis, Luke D; Betzig, Eric; Tjian, Robert; Liu, Zhe

2016-01-01

The presumptive altered dynamics of transient molecular interactions in vivo contributing to neurodegenerative diseases have remained elusive. Here, using single-molecule localization microscopy, we show that disease-inducing Huntingtin (mHtt) protein fragments display three distinct dynamic states in living cells – 1) fast diffusion, 2) dynamic clustering and 3) stable aggregation. Large, stable aggregates of mHtt exclude chromatin and form 'sticky' decoy traps that impede target search processes of key regulators involved in neurological disorders. Functional domain mapping based on super-resolution imaging reveals an unexpected role of aromatic amino acids in promoting protein-mHtt aggregate interactions. Genome-wide expression analysis and numerical simulation experiments suggest mHtt aggregates reduce transcription factor target site sampling frequency and impair critical gene expression programs in striatal neurons. Together, our results provide insights into how mHtt dynamically forms aggregates and disrupts the finely-balanced gene control mechanisms in neuronal cells. DOI: http://dx.doi.org/10.7554/eLife.17056.001 PMID:27484239

Three-dimensional Myoblast Aggregates--Effects of Modeled Microgravity

NASA Technical Reports Server (NTRS)

Byerly, Diane; Sognier, M. A.; Marquette, M. L.

2006-01-01

The overall objective of these studies is to elucidate the molecular and cellular alterations that contribute to muscle atrophy in astronauts caused by exposure to microgravity conditions in space. To accomplish this, a three-dimensional model test system was developed using mouse myoblast cells (C2C12). Myoblast cells were grown as three-dimensional aggregates (without scaffolding or other solid support structures) in both modeled microgravity (Rotary Cell Culture System, Synthecon, Inc.) and at unit gravity in coated Petri dishes. Evaluation of H&E stained thin sections of the aggregates revealed the absence of any necrosis. Confocal microscopy evaluations of cells stained with the Live/Dead assay (Molecular Probes) confirmed that viable cells were present throughout the aggregates with an average of only three dead cells observed per aggregate. Preliminary results from gene array analysis (Affymetrix chip U74Av2) showed that approximately 14% of the genes were down regulated (decreased more than 3 fold) and 4% were upregulated in cells exposed to modeled microgravity for 12 hours compared to unit gravity controls. Additional studies using fluorescent phallacidin revealed a decrease in F-actin in the cells exposed to modeled microgravity compared to unit gravity. Myoblast cells grown as aggregates in modeled microgravity exhibited spontaneous differentiation into syncitia while no differentiation was seen in the unit gravity controls. These studies show that 1)the model test system developed is suitable for assessing cellular and molecular alterations in myoblasts; 2) gene expression alterations occur rapidly (within 12 hours) following exposure to modeled microgravity; and 3) modeled microgravity conditions stimulated myoblast cell differentiation. Achieving a greater understanding of the molecular alterations leading to muscle atrophy will eventually enable the development of cell-based countermeasures, which may be valuable for treatment of muscle diseases on Earth and future space explorations.

Non-cell autonomous cell death caused by transmission of Huntingtin aggregates in Drosophila.

PubMed

Babcock, Daniel T; Ganetzky, Barry

2015-01-01

Recent evidence indicates that protein aggregates can spread between neurons in several neurodegenerative diseases but much remains unknown regarding the underlying mechanisms responsible for this spreading and its role in disease progression. We recently demonstrated that mutant Huntingtin aggregates spread between cells within the Drosophila brain resulting in non-cell autonomous loss of a pair of large neurons in the posterior protocerebrum. However, the full extent of neuronal loss throughout the brain was not determined. Here we examine the effects of driving expression of mutant Huntingtin in Olfactory Receptor Neurons (ORNs) by using a marker for cleaved caspase activity to monitor neuronal apoptosis as a function of age. We find widespread caspase activity in various brain regions over time, demonstrating that non-cell autonomous damage is widespread. Improved understanding of which neurons are most vulnerable and why should be useful in developing treatment strategies for neurodegenerative diseases that involve transcellular spreading of aggregates.

Apple Procyanidins Suppress Amyloid β-Protein Aggregation

PubMed Central

Toda, Toshihiko; Sunagawa, Tadahiro; Kanda, Tomomasa; Tagashira, Motoyuki; Shirasawa, Takuji; Shimizu, Takahiko

2011-01-01

Procyanidins (PCs) are major components of the apple polyphenols (APs). We previously reported that treatment with PC extended the mean lifespan of Caenorhabditis elegans (Sunagawa et al., 2011). In order to estimate the neuroprotective effects of PC, we investigated the antiaggregative activity of PC on amyloid β-protein (Aβ) aggregation, which is a pathological hallmark of Alzheimer's disease. We herein report that PC significantly suppressed Aβ42 aggregation and dissociated Aβ42 aggregates in a dose-dependent manner, indicating that PC is a potent suppressor of Aβ aggregation. Furthermore, PC significantly inhibited Aβ42 neurotoxicity and stimulated proliferation in PC-12 cells. These results suggested that the PC and AP acted as neuroprotective factors against toxic Aβ aggregates. PMID:21826271

Competitive aggregation dynamics using phase wave signals.

PubMed

Sakaguchi, Hidetsugu; Maeyama, Satomi

2014-10-21

Coupled equations of the phase equation and the equation of cell concentration n are proposed for competitive aggregation dynamics of slime mold in two dimensions. Phase waves are used as tactic signals of aggregation in this model. Several aggregation clusters are formed initially, and target patterns appear around the localized aggregation clusters. Owing to the competition among target patterns, the number of the localized aggregation clusters decreases, and finally one dominant localized pattern survives. If the phase equation is replaced with the complex Ginzburg-Landau equation, several spiral patterns appear, and n is localized near the center of the spiral patterns. After the competition among spiral patterns, one dominant spiral survives. Copyright © 2014 Elsevier Ltd. All rights reserved.

Mechanical Dissociation of Platelet Aggregates in Blood Stream

NASA Astrophysics Data System (ADS)

Hoore, Masoud; Fedosov, Dmitry A.; Gompper, Gerhard; Complex; Biological Fluids Group Team

2017-11-01

von Willebrand factor (VWF) and platelet aggregation is a key phenomenon in blood clotting. These aggregates form critically in high shear rates and dissolve reversibly in low shear rates. The emergence of a critical shear rate, beyond which aggregates form and below which they dissolve, has an interesting impact on aggregation in blood flow. As red blood cells (RBCs) migrate to the center of the vessel in blood flow, a RBC free layer (RBC-FL) is left close to the walls into which the platelets and VWFs are pushed back from the bulk flow. This margination process provides maximal VWF-platelet aggregation probability in the RBC-FL. Using mesoscale hydrodynamic simulations of aggregate dynamics in blood flow, it is shown that the aggregates form and grow in RBC-FL wherein shear rate is high for VWF stretching. By growing, the aggregates penetrate to the bulk flow and get under order of magnitude lower shear rates. Consequently, they dissolve and get back into the RBC-FL. This mechanical limitation for aggregates prohibits undesired thrombosis and vessel blockage by aggregates, while letting the VWFs and platelets to aggregate close to the walls where they are actually needed. The support by the DFG Research Unit FOR 1543 SHENC and CPU time Grant by the Julich Supercomputing Center are acknowledged.

Theoretical deposition of carcinogenic particle aggregates in the upper respiratory tract.

PubMed

Sturm, Robert

2013-10-01

Numerous particles suspended in the atmosphere are composed of smaller particular components that form aggregates with highly irregular shape. Such aggregates, among which dusts and soot are the most prominent examples, may be taken up into the respiratory tract and, in the worst case, initiate a malignant transformation of lung cells. Particle aggregates were theoretically modelled by using small spheres with equal diameters (1 nm) and arranging them randomly. This procedure resulted in the generation of various aggregate shapes (chain-like, loose, compact), for which essential parameters such as dynamic shape factors, χ, and aerodynamic diameters, dae , were computed. Deposition of aggregates consisting of 10, 50, 100, and 1,000 nano-spheres was simulated for the uppermost parts of the human respiratory system (extrathoracic region and airway generation 0 to 4), thereby distinguishing between sitting and light-work breathing as well as between nasal and oral inhalation. Based upon the modelling results, aggregate deposition in the human respiratory system can be described as a function of (I) aerodynamic diameter; (II) inhaled particle position within the airway system; and (III) breathing conditions. Therefore, highest deposition values were obtained for nano-scale aggregates (<10 nm), whereas larger aggregates exhibited slightly to significantly reduced deposition probabilities. Extrathoracic regions and uppermost bronchi (generations 0 to 1) were marked by most effective particle capture. Any increase of inhaled air volumes and reduction of breathing times resulted in an enhancement of deposition probabilities of larger particles. Based on the results derived from this study it may be concluded that small particle aggregates are accumulated in the uppermost compartments of the human respiratory tract, where they may unfold their unwholesome potential. In the case of carcinogenic particles being stored in epithelial cells for a longer time span, malignant transformations starting with the formation of cancerous cells and ending with the growth of a tumour have to be assumed.

Influence of plankton community structure on the sinking velocity of marine aggregates

NASA Astrophysics Data System (ADS)

Bach, L. T.; Boxhammer, T.; Larsen, A.; Hildebrandt, N.; Schulz, K. G.; Riebesell, U.

2016-08-01

About 50 Gt of carbon is fixed photosynthetically by surface ocean phytoplankton communities every year. Part of this organic matter is reprocessed within the plankton community to form aggregates which eventually sink and export carbon into the deep ocean. The fraction of organic matter leaving the surface ocean is partly dependent on aggregate sinking velocity which accelerates with increasing aggregate size and density, where the latter is controlled by ballast load and aggregate porosity. In May 2011, we moored nine 25 m deep mesocosms in a Norwegian fjord to assess on a daily basis how plankton community structure affects material properties and sinking velocities of aggregates (Ø 80-400 µm) collected in the mesocosms' sediment traps. We noted that sinking velocity was not necessarily accelerated by opal ballast during diatom blooms, which could be due to relatively high porosity of these rather fresh aggregates. Furthermore, estimated aggregate porosity (Pestimated) decreased as the picoautotroph (0.2-2 µm) fraction of the phytoplankton biomass increased. Thus, picoautotroph-dominated communities may be indicative for food webs promoting a high degree of aggregate repackaging with potential for accelerated sinking. Blooms of the coccolithophore Emiliania huxleyi revealed that cell concentrations of 1500 cells/mL accelerate sinking by about 35-40%, which we estimate (by one-dimensional modeling) to elevate organic matter transfer efficiency through the mesopelagic from 14 to 24%. Our results indicate that sinking velocities are influenced by the complex interplay between the availability of ballast minerals and aggregate packaging; both of which are controlled by plankton community structure.

Mathematical model of macrophage-facilitated breast cancer cells invasion.

PubMed

Knútsdóttir, Hildur; Pálsson, Eirikur; Edelstein-Keshet, Leah

2014-09-21

Mortality from breast cancer stems from its tendency to invade into surrounding tissues and organs. Experiments have shown that this metastatic process is facilitated by macrophages in a short-ranged chemical signalling loop. Macrophages secrete epidermal growth factor, EGF, and respond to the colony stimulating factor 1, CSF-1. Tumor cells secrete CSF-1 and respond to EGF. In this way, the cells coordinate aggregation and cooperative migration. Here we investigate this process in a model for in vitro interactions using two distinct but related mathematical approaches. In the first, we analyze and simulate a set of partial differential equations to determine conditions for aggregation. In the second, we use a cell-based discrete 3D simulation to follow the fates and motion of individual cells during aggregation. Linear stability analysis of the PDE model reveals that decreasing the chemical secretion, chemotaxis coefficients or density of cells or increasing the chemical degradation in the model could eliminate the spontaneous aggregation of cells. Simulations with the discrete model show that the ratio between tumor cells and macrophages in aggregates increases when the EGF secretion parameter is increased. The results also show how CSF-1/CSF-1R autocrine signalling in tumor cells affects the ratio between the two cell types. Comparing the continuum results with simulations of a discrete cell-based model, we find good qualitative agreement. Copyright © 2014 Elsevier Ltd. All rights reserved.

Cell and organ printing 2: fusion of cell aggregates in three-dimensional gels.

PubMed

Boland, Thomas; Mironov, Vladimir; Gutowska, Anna; Roth, Elisabeth A; Markwald, Roger R

2003-06-01

We recently developed a cell printer (Wilson and Boland, 2003) that enables us to place cells in positions that mimic their respective positions in organs. However, this technology was limited to the printing of two-dimensional (2D) tissue constructs. Here we describe the use of thermosensitive gels to generate sequential layers for cell printing. The ability to drop cells on previously printed successive layers provides a real opportunity for the realization of three-dimensional (3D) organ printing. Organ printing will allow us to print complex 3D organs with computer-controlled, exact placing of different cell types, by a process that can be completed in several minutes. To demonstrate the feasibility of this novel technology, we showed that cell aggregates can be placed in the sequential layers of 3D gels close enough for fusion to occur. We estimated the optimum minimal thickness of the gel that can be reproducibly generated by dropping the liquid at room temperature onto a heated substrate. Then we generated cell aggregates with the corresponding (to the minimal thickness of the gel) size to ensure a direct contact between printed cell aggregates during sequential printing cycles. Finally, we demonstrated that these closely-placed cell aggregates could fuse in two types of thermosensitive 3D gels. Taken together, these data strongly support the feasibility of the proposed novel organ-printing technology. Copyright 2003 Wiley-Liss, Inc.

Genetics Home Reference: spinocerebellar ataxia type 1

MedlinePlus

... Within cells, ataxin-1 is located in the nucleus . Researchers believe that ataxin-1 may be involved ... other proteins to form clumps (aggregates) within the nucleus of the cells. These aggregates prevent the ataxin- ...

A Theoretical Study of some Rheological Properties of the Aggregation of the Molecules Deoxy- Hemoglobin S

NASA Astrophysics Data System (ADS)

Mensah, Francis; Grant, Julius; Thorpe, Arthur

2010-02-01

Sickle cell disease is a serious public health problem that affects many people worldwide. In this paper, the Langevin equation is used for hemoglobin's aggregation in sickle cell anemia. Several parameters are explored such as the time-dependent deformation of the aggregates whose plot gives a sigmoid, the time-dependent expressions obtained for the coefficient of viscosity and the elastic modulus which characterize the aggregation of the sickle hemoglobin. Other properties such as the viscoelastic and the elasto-thixotropic properties of the sickle hemoglobin polymer are also described. An attempt is made to approach the polymerization process in terms of a dynamical system. )

Aggregation of the rhizospheric bacterium Azospirillum brasilense in response to oxygen

NASA Astrophysics Data System (ADS)

Abdoun, Hamid; McMillan, Mary; Pereg, Lily

2016-04-01

Azospirillum brasilense spp. have ecological, scientific and agricultural importance. As model plant growth promoting rhizobacteria they interact with a large variety of plants, including important food and cash crops. Azospirillum strains are known for their production of plant growth hormones that enhance root systems and for their ability to fix nitrogen. Azospirillum cells transform in response to environmental cues. The production of exopolysaccharides and cell aggregation during cellular transformation are important steps in the attachment of Azospirillum to roots. We investigate signals that induce cellular transformation and aggregation in the Azospirillum and report on the importance of oxygen to the process of aggregation in this rhizospheric bacterium.

Universal timescales in the rheology of spheroid cell aggregates

NASA Astrophysics Data System (ADS)

Yu, Miao; Mahtabfar, Aria; Beleen, Paul; Foty, Ramsey; Zahn, Jeffrey; Shreiber, David; Liu, Liping; Lin, Hao

2017-11-01

The rheological properties of tissue play important roles in key biological processes including embryogenesis, cancer metastasis, and wound healing. Spheroid cell aggregate is a particularly interesting model system for the study of these phenomena. In the long time, they behave like drops with a surface tension. In the short, viscoelasticity also needs to be considered. In this work, we discover two coupled and universal timescales for spheroid aggregates. A total of 12 aggregate types (total aggregate number n =290) derived from L and GBM (glioblastoma multiforme) cells are studied with microtensiometer to obtain their surface tension. They are also allowed to relax upon release of the compression forces. The two timescales are observed during the relaxation process; their values do not depend on compression time nor the degree of deformation, and are consistent among all 12 types. Following prior work (Yu et al., Phys. Rev. Lett., 115:128303; Liu et al., J. Mech. Phys. Solids, 98:309-329) we use a rigorous mathematical theory to interpret the results, which reveals intriguing properties of the aggregates on both tissue and cellular levels. The mechanics of multicellular organization reflects both complexity and regularity due to strong active regulation.

Three-dimensional printed polymeric system to encapsulate human mesenchymal stem cells differentiated into islet-like insulin-producing aggregates for diabetes treatment.

PubMed

Sabek, Omaima M; Farina, Marco; Fraga, Daniel W; Afshar, Solmaz; Ballerini, Andrea; Filgueira, Carly S; Thekkedath, Usha R; Grattoni, Alessandro; Gaber, A Osama

2016-01-01

Diabetes is one of the most prevalent, costly, and debilitating diseases in the world. Pancreas and islet transplants have shown success in re-establishing glucose control and reversing diabetic complications. However, both are limited by donor availability, need for continuous immunosuppression, loss of transplanted tissue due to dispersion, and lack of vascularization. To overcome the limitations of poor islet availability, here, we investigate the potential of bone marrow-derived mesenchymal stem cells differentiated into islet-like insulin-producing aggregates. Islet-like insulin-producing aggregates, characterized by gene expression, are shown to be similar to pancreatic islets and display positive immunostaining for insulin and glucagon. To address the limits of current encapsulation systems, we developed a novel three-dimensional printed, scalable, and potentially refillable polymeric construct (nanogland) to support islet-like insulin-producing aggregates' survival and function in the host body. In vitro studies showed that encapsulated islet-like insulin-producing aggregates maintained viability and function, producing steady levels of insulin for at least 4 weeks. Nanogland-islet-like insulin-producing aggregate technology here investigated as a proof of concept holds potential as an effective and innovative approach for diabetes cell therapy.

The common inhaled anesthetic isoflurane increases aggregation of huntingtin and alters calcium homeostasis in a cell model of Huntington's disease

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

Wang Qiujun; Department of Anesthesiology, The Third Clinical Hospital, Hebei Medical University, Shijiazhuang, Hebei 050051; Liang Ge

2011-02-01

Isoflurane is known to increase {beta}-amyloid aggregation and neuronal damage. We hypothesized that isoflurane will have similar effects on the polyglutamine huntingtin protein and will cause alterations in intracellular calcium homeostasis. We tested this hypothesis in striatal cells from the expanded glutamine huntingtin knock-in mouse (STHdh{sup Q111/Q111}) and wild type (STHdh{sup Q7/Q7}) striatal neurons. The primary cultured neurons were exposed for 24 h to equipotent concentrations of isoflurane, sevoflurane, and desflurane in the presence or absence of extracellular calcium and with or without xestospongin C, a potent endoplasmic reticulum inositol 1,4,5-trisphosphate (InsP{sub 3}) receptor antagonist. Aggregation of huntingtin protein, cellmore » viability, and calcium concentrations were measured. Isoflurane, sevoflurane, and desflurane all increased the aggregation of huntingtin in STHdh{sup Q111/Q111} cells, with isoflurane having the largest effect. Isoflurane induced greater calcium release from the ER and relatively more cell damage in the STHdh{sup Q111/Q111} huntingtin cells than in the wild type STHdh{sup Q7/Q7} striatal cells. However, sevoflurane and desflurane caused less calcium release from the ER and less cell damage. Xestospongin C inhibited the isoflurane-induced calcium release from the ER, aggregation of huntingtin, and cell damage in the STHdh{sup Q111/Q111} cells. In summary, the Q111 form of huntingtin increases the vulnerability of striatal neurons to isoflurane neurotoxicity through combined actions on the ER IP{sub 3} receptors. Calcium release from the ER contributes to the anesthetic induced huntingtin aggregation in STHdh{sup Q111/Q111} striatal cells.« less

Intracellular growth of Mycobacterium tuberculosis after macrophage cell death leads to serial killing of host cells

PubMed Central

Mahamed, Deeqa; Boulle, Mikael; Ganga, Yashica; Mc Arthur, Chanelle; Skroch, Steven; Oom, Lance; Catinas, Oana; Pillay, Kelly; Naicker, Myshnee; Rampersad, Sanisha; Mathonsi, Colisile; Hunter, Jessica; Wong, Emily B; Suleman, Moosa; Sreejit, Gopalkrishna; Pym, Alexander S; Lustig, Gila; Sigal, Alex

2017-01-01

A hallmark of pulmonary tuberculosis is the formation of macrophage-rich granulomas. These may restrict Mycobacterium tuberculosis (Mtb) growth, or progress to central necrosis and cavitation, facilitating pathogen growth. To determine factors leading to Mtb proliferation and host cell death, we used live cell imaging to track Mtb infection outcomes in individual primary human macrophages. Internalization of Mtb aggregates caused macrophage death, and phagocytosis of large aggregates was more cytotoxic than multiple small aggregates containing similar numbers of bacilli. Macrophage death did not result in clearance of Mtb. Rather, it led to accelerated intracellular Mtb growth regardless of prior activation or macrophage type. In contrast, bacillary replication was controlled in live phagocytes. Mtb grew as a clump in dead cells, and macrophages which internalized dead infected cells were very likely to die themselves, leading to a cell death cascade. This demonstrates how pathogen virulence can be achieved through numbers and aggregation states. DOI: http://dx.doi.org/10.7554/eLife.22028.001 PMID:28130921

Shortening of the Lactobacillus paracasei subsp. paracasei BGNJ1-64 AggLb Protein Switches Its Activity from Auto-aggregation to Biofilm Formation.

PubMed

Miljkovic, Marija; Bertani, Iris; Fira, Djordje; Jovcic, Branko; Novovic, Katarina; Venturi, Vittorio; Kojic, Milan

2016-01-01

AggLb is the largest (318.6 kDa) aggregation-promoting protein of Lactobacillus paracasei subsp. paracasei BGNJ1-64 responsible for forming large cell aggregates, which causes auto-aggregation, collagen binding and pathogen exclusion in vitro. It contains an N-terminus leader peptide, followed by six successive collagen binding domains, 20 successive repeats (CnaB-like domains) and an LPXTG sorting signal at the C-terminus for cell wall anchoring. Experimental information about the roles of the domains of AggLb is currently unknown. To define the domain that confers cell aggregation and the key domains for interactions of specific affinity between AggLb and components of the extracellular matrix, we constructed a series of variants of the aggLb gene and expressed them in Lactococcus lactis subsp. lactis BGKP1-20 using a lactococcal promoter. All of the variants contained a leader peptide, an inter collagen binding-CnaB domain region (used to raise an anti-AggLb antibody), an anchor domain and a different number of collagen binding and CnaB-like domains. The role of the collagen binding repeats of the N-terminus in auto-aggregation and binding to collagen and fibronectin was confirmed. Deletion of the collagen binding repeats II, III, and IV resulted in a loss of the strong auto-aggregation, collagen and fibronectin binding abilities whereas the biofilm formation capability was increased. The strong auto-aggregation, collagen and fibronectin binding abilities of AggLb were negatively correlated to biofilm formation.

Engineering cell aggregates through incorporated polymeric microparticles.

PubMed

Ahrens, Caroline C; Dong, Ziye; Li, Wei

2017-10-15

Ex vivo cell aggregates must overcome significant limitations in the transport of nutrients, drugs, and signaling proteins compared to vascularized native tissue. Further, engineered extracellular environments often fail to sufficiently replicate tethered signaling cues and the complex architecture of native tissue. Co-cultures of cells with microparticles (MPs) is a growing field directed towards overcoming many of these challenges by providing local and controlled presentation of both soluble and tethered proteins and small molecules. Further, co-cultured MPs offer a mechanism to better control aggregate architecture and even to report key characteristics of the local microenvironment such as pH or oxygen levels. Herein, we provide a brief introduction to established and developing strategies for MP production including the choice of MP materials, fabrication techniques, and techniques for incorporating additional functionality. In all cases, we emphasize the specific utility of each approach to form MPs useful for applications in cell aggregate co-culture. We review established techniques to integrate cells and MPs. We highlight those strategies that promote targeted heterogeneity or homogeneity, and we describe approaches to engineer cell-particle and particle-particle interactions that enhance aggregate stability and biological response. Finally, we review advances in key application areas of MP aggregates and future areas of development. Cell-scaled polymer microparticles (MPs) integrated into cellular aggregates have been shown to be a powerful tool to direct cell response. MPs have supported the development of healthy cartilage, islets, nerves, and vasculature by the maintenance of soluble gradients as well as by the local presentation of tethered cues and diffusing proteins and small molecules. MPs integrated with pluripotent stem cells have directed in vivo expansion and differentiation. Looking forward, MPs are expected to support both the characterization and development of in vitro tissue systems for applications such as drug testing platforms. However, useful co-cultures must be designed keeping in mind the limitations and attributes of each material strategy within the context of the overall tissue biology. The present review integrates prospectives from materials development, drug delivery, and tissue engineering to provide a toolbox for the development and application of MPs useful for long-term co-culture within cell aggregates. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Are there multiple pathways in the pathogenesis of Huntington's disease?

PubMed Central

Aronin, N; Kim, M; Laforet, G; DiFiglia, M

1999-01-01

Studies of huntingtin localization in human post-mortem brain offer insights and a framework for basic experiments in the pathogenesis of Huntington's disease. In neurons of cortex and striatum, we identified changes in the cytoplasmic localization of huntingtin including a marked perinuclear accumulation of huntingtin and formation of multivesicular bodies, changes conceivably pointing to an altered handling of huntingtin in neurons. In Huntington's disease, huntingtin also accumulates in aberrant subcellular compartments such as nuclear and neuritic aggregates co-localized with ubiquitin. The site of protein aggregation is polyglutamine-dependent, both in juvenile-onset patients having more aggregates in the nucleus and in adult-onset patients presenting more neuritic aggregates. Studies in vitro reveal that the genesis of these aggregates and cell death are tied to cleavage of mutant huntingtin. However, we found that the aggregation of mutant huntingtin can be dissociated from the extent of cell death. Thus properties of mutant huntingtin more subtle than its aggregation, such as its proteolysis and protein interactions that affect vesicle trafficking and nuclear transport, might suffice to cause neurodegeneration in the striatum and cortex. We propose that mutant huntingtin engages multiple pathogenic pathways leading to neuronal death. PMID:10434298

Protein carbonylation, protein aggregation and neuronal cell death in a murine model of multiple sclerosis

NASA Astrophysics Data System (ADS)

Dasgupta, Anushka

Many studies have suggested that oxidative stress plays an important role in the pathophysiology of both multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). Yet, the mechanism by which oxidative stress leads to tissue damage in these disorders is unclear. Recent work from our laboratory has revealed that protein carbonylation, a major oxidative modification caused by severe and/or chronic oxidative stress conditions, is elevated in MS and EAE. Furthermore, protein carbonylation has been shown to alter protein structure leading to misfolding/aggregation. These findings prompted me to hypothesize that carbonylated proteins, formed as a consequence of oxidative stress and/or decreased proteasomal activity, promote protein aggregation to mediate neuronal apoptosis in vitro and in EAE. To test this novel hypothesis, I first characterized protein carbonylation, protein aggregation and apoptosis along the spinal cord during the course of myelin-oligodendrocyte glycoprotein (MOG)35-55 peptide-induced EAE in C57BL/6 mice [Chapter 2]. The results show that carbonylated proteins accumulate throughout the course of the disease, albeit by different mechanisms: increased oxidative stress in acute EAE and decreased proteasomal activity in chronic EAE. I discovered not only that there is a temporal correlation between protein carbonylation and apoptosis but also that carbonyl levels are significantly higher in apoptotic cells. A high number of juxta-nuclear and cytoplasmic protein aggregates containing the majority of the oxidized proteins are also present during the course of EAE, which seems to be due to reduced autophagy. In chapter 3, I show that when gluthathione levels are reduced to those in EAE spinal cord, both neuron-like PC12 (nPC12) cells and primary neuronal cultures accumulate carbonylated proteins and undergo cell death (both by necrosis and apoptosis). Immunocytochemical and biochemical studies also revealed a temporal/spatial relationship between carbonylation, protein aggregation and cellular apoptosis. Furthermore, the effectiveness of the carbonyl scavenger hydralazine, histidine hydrazide and methoxylamine at preventing cell death identifies protein carbonyls as the toxic species. Experiments using well-characterized apoptosis inhibitors place protein carbonylation downstream of the mitochondrial transition pore opening and upstream of caspase activation. These in vitro studies demonstrate for the first time a causal relationship between carbonylation, protein aggregation and apoptosis of neurons undergoing oxidative damage. This relationship was further strengthened with the experiments carried out in chapter 4, which show that inhibition of protein aggregation with congo red (CR) or 2-hydroxypropyl beta-cyclodextrin (HPCD) significantly reduced neuronal cell death without affecting the levels of oxidized proteins. Interestingly, large, juxta-nuclear aggregates are not formed upon GSH depletion, suggesting that the small protein aggregates are the cytotoxic species. Together, our data suggest that protein carbonylation causes protein aggregation to mediate neuronal apoptosis in vitro and that a similar mechanism might be contributing to neuronal/glial apoptosis in EAE. These studies provide the basis for testing protein carbonylation scavengers and protein aggregation inhibitors for the treatment of inflammatory demyelinating disorders.

First haemorheological experiment on NASA space shuttle 'Discovery' STS 51-C: aggregation of red cells.

PubMed

Dintenfass, L; Osman, P D; Jedrzejczyk, H

1985-01-01

The 'secret' D.O.D. Mission on flight STS 51-C also carried nearly 100 kg of automated instrumentation of the Australian experiment on aggregation of red cells ("ARC"). The automated Slit-Capillary Photo Viscometer contained blood samples from subjects with history of coronary heart disease, cancer of the colon, insulin-dependent diabetes, etc., as well as normals. The experiment ran for nine hours, according to the program of its microcomputers. When shuttle landed and instrumentation recovered and opened in the presence of NASA quality control officers, it was obvious that experiment was a success. Tentative and preliminary results can be summarized as follows: red cells did not change shape under zero gravity; red cells do aggregate under zero gravity, although the size of aggregates is smaller than on the ground; the morphology of aggregates of red cells appears to be of rouleaux type under zero gravity, notwithstanding the fact that pathological blood was used. These results will have to be confirmed in the future flights. The background and history of development of the project are described, and put into context of our general haemorheological studies.

The aggregational status of cholera enterotoxin fragment A following biochemical fractionation.

PubMed

Knoop, F C

1978-01-01

Aggregates of frabment A of Vibrio cholerae enterotoxin were revealed following isoelectric focusing in 8 M urea of molecular sieve chromatography in 4% (v/v) formic acid. These aggregates consisted of dimers which required the presence of 10 M urea, 1% sodium dodecyl sulfate (SDS), 2 mM ethylenediaminetetracetic acid (EDTA) and heat (60 degrees C for 1 h) for complete dissociation. All aggregates were homogeneous when tested by standard analytical and SDS polyacrylamide gel electrophoresis or immunodiffusion analysis. Aggregates of fragment A were biologically active in the mouse Y1 adrenal cell assay.

Dispatch. Dictyostelium chemotaxis: fascism through the back door?

PubMed

Insall, Robert

2003-04-29

Aggregating Dictyostelium cells secrete cyclic AMP to attract their neighbours by chemotaxis. It has now been shown that adenylyl cyclase is enriched in the rear of cells, and this localisation is required for normal aggregation.

A Study of Blood Flow and of Aggregation of Blood Cells Under Conditions of Zero Gravity: Its Relevance to the Occlusive Diseases and Cancer

NASA Technical Reports Server (NTRS)

Dintenfass, L.

1985-01-01

The objectives of this program are: (1) to determine whether the size of red cell aggregates, kinetics and morphology of these aggregates are influenced by near-zero gravity; (2) whether viscosity, especially at low shear rate, is afflicted by near-zero gravity (the latter preventing sedimentation of red cells); (3) whether the actual shape of red cells changes; and (4) whether blood samples obtained from different donors (normal and patients suffering from different disorders) react in the same manner to near-zero gravity.

Improving the quality of miniature pig somatic cell nuclear transfer blastocysts: aggregation of SCNT embryos at the four-cell stage.

PubMed

Terashita, Y; Sugimura, S; Kudo, Y; Amano, R; Hiradate, Y; Sato, E

2011-04-01

Miniature pigs share many similar characteristics such as anatomy, physiology and body size with humans and are expected to become important animal models for therapeutic cloning using embryonic stem cells (ESCs) derived by somatic cell nuclear transfer (SCNT). In the present study, we observed that miniature pig SCNT blastocysts possessed a lower total number of nuclei and a lower percentage of POU5F1-positive cells than those possessed by in vitro fertilized (IVF) blastocysts. To overcome these problems, we evaluated the applicability of aggregating miniature pig SCNT embryos at the four-cell stage. We showed that (i) aggregation of two or three miniature pig SCNT embryos at the four-cell stage improves the total number of nuclei and the percentage of POU5F1-positive cells in blastocysts, and (ii) IVF blastocysts with low cell numbers induced by the removal of two blastomeres at the four-cell stage did not exhibit a decrease in the percentage of POU5F1-positive cells. These results suggest that the aggregation of miniature pig SCNT embryos at the four-cell stage can be a useful technique for improving the quality of miniature pig SCNT blastocysts and indicating that improvement in the percentage of POU5F1-positive cells in aggregated SCNT embryos is not simply the consequence of increased cell numbers. © 2010 Blackwell Verlag GmbH.

No tillage effect on water retention characteristics of soil aggregates in rainfed semiarid conditions.

NASA Astrophysics Data System (ADS)

Blanco-Moure, Nuria; López, M. Victoria; Moret, David

2010-05-01

The evaluation of changes in soil moisture retention characteristics associated to alterations in soil structure is of great interest in tillage studies. Most of these studies have evaluated soil properties in samples of total soil but not in individual aggregates. However, soil behavior at a macroscale level depends on the aggregate properties. A better knowledge of aggregate characteristics, as the water retention properties, will help to explain, for example, the response of soil to tillage, compaction and crop growth, and hence, to plan adequate soil management practices. In this study we determine the water retention curve of soil aggregates of different sizes from a soil under two tillage systems (conventional and no tillage). The study was carried out in a silty clay loam soil of semiarid Aragon (NE Spain). Two tillage systems were compared: no tillage (NT) and conventional tillage with mouldboard plough (CT). Water retention curves (WRC) were determined for soil surface aggregates (0-5 cm) of three different sizes (8-4, 4-2 and 2-1 mm in diameter) by using the TDR-pressure cell (Moret et al. 2008. Soil Till. Res, 100, 114-119). The TDR-pressure cell is a non-destructive method which permits determining WRC with the only one and same soil sample. Thus, the pressure cell was filled with aggregates up to 4 cm height, weighted and wetted to saturation from the bottom. Pressure steps were sequentially applied at -0.5, -1.5, -3, -5, -10, -33, -100, -300 kPa, and water content of each aggregate sample was measured gravimetrically and by TDR 24 h after starting each pressure head step. The volume of the sample within the cell was also determined at this moment in order to obtain the bulk density and thus calculate the volumetric water content. A good relationship was obtained between the volumetric water content calculated from the gravimetric water content and the corresponding values measured by TDR (r2=0.907; p≤0.05). Within the same tillage treatment, no significant differences in WRC were found among soil aggregate sizes. Soil aggregates under CT retained more water at lower pressure heads in all aggregate sizes; in contrast the retention was more effective in those from NT at high pressure level. The extensive structural degradation of the CT aggregates observed during wetting with the consequent decrease in the soil volume within the transparent cell, can help to explain the different behaviour of both soils. The CT aggregates were probably disintegrated by slaking, causing a reduction in water drainage and, therefore, an increase in soil water content at low pressure heads. This idea was also confirmed with the application of the double exponential function proposed by Dexter et al. (2008. Geoderma 173, 243-253). The WRC curves measured by TDR were successfully fitted to the theoretical model proposed by Dexter (r2=0.986; p≤0.05). Thus, the model estimated that the large porosity between aggregates retain slightly more water under CT (0.36-0.39 m3 m-3) than under NT (0.31-0.35 m3 m-3). On the contrary, pores inside the aggregates tend to storage more water in NT (0.16-0.20 m3 m-3vs. 0.13-0.17 m3 m-3 in CT). These results show the suitability of NT to reduce the risk of soil crusting and compaction in agricultural lands of Aragón.

Reduced receptor aggregation and altered cytoskeleton in cultured myocytes after space-flight

NASA Technical Reports Server (NTRS)

Gruener, R.; Roberts, R.; Reitstetter, R.

1994-01-01

We carried out parallel experiments first on the slow clinostat and then in space-flight to examine the effects of altered gravity on the aggregation of the nicotinic acetylcholine receptors and the structure of the cytoskeleton in cultured Xenopus embryonic muscle cells. By examining the concordance between results from space flight and the clinostat, we tested whether the slow clinostat is a relevant simulation paradigm. Space-flown cells showed marked changes in the distribution and organization of actin filaments and had a reduced incidence of acetylcholine receptor aggregates at the site of contact with polystyrene beads. Similar effects were found after clinostat rotation. The sensitivity of synaptic receptor aggregation and cytoskeletal morphology suggests that in the microgravity of space cell behavior may be importantly altered.

Bistable Expression of CsgD in Salmonella enterica Serovar Typhimurium Connects Virulence to Persistence

PubMed Central

MacKenzie, Keith D.; Wang, Yejun; Shivak, Dylan J.; Wong, Cynthia S.; Hoffman, Leia J. L.; Lam, Shirley; Kröger, Carsten; Cameron, Andrew D. S.; Townsend, Hugh G. G.; Köster, Wolfgang

2015-01-01

Pathogenic bacteria often need to survive in the host and the environment, and it is not well understood how cells transition between these equally challenging situations. For the human and animal pathogen Salmonella enterica serovar Typhimurium, biofilm formation is correlated with persistence outside a host, but the connection to virulence is unknown. In this study, we analyzed multicellular-aggregate and planktonic-cell subpopulations that coexist when S. Typhimurium is grown under biofilm-inducing conditions. These cell types arise due to bistable expression of CsgD, the central biofilm regulator. Despite being exposed to the same stresses, the two cell subpopulations had 1,856 genes that were differentially expressed, as determined by transcriptome sequencing (RNA-seq). Aggregated cells displayed the characteristic gene expression of biofilms, whereas planktonic cells had enhanced expression of numerous virulence genes. Increased type three secretion synthesis in planktonic cells correlated with enhanced invasion of a human intestinal cell line and significantly increased virulence in mice compared to the aggregates. However, when the same groups of cells were exposed to desiccation, the aggregates survived better, and the competitive advantage of planktonic cells was lost. We hypothesize that CsgD-based differentiation is a form of bet hedging, with single cells primed for host cell invasion and aggregated cells adapted for persistence in the environment. This allows S. Typhimurium to spread the risks of transmission and ensures a smooth transition between the host and the environment. PMID:25824832

Ethanol does not inhibit the adhesive activity of Drosophila neuroglian or human L1 in Drosophila S2 tissue culture cells.

PubMed

Vallejo, Y; Hortsch, M; Dubreuil, R R

1997-05-02

Members of the L1 family of homophilic neural cell adhesion molecules are thought to play an important role in nervous system development and function. It is also suggested that L1 is a direct target of ethanol in fetal alcohol syndrome, since ethanol inhibits the aggregation of cultured cells expressing L1 (Ramanathan, R., Wilkemeyer, M. F., Mittel, B., Perides, G., and Charness, M. E. (1996) J. Cell Biol. 133, 381-390). If ethanol acts directly on the homophilic adhesive function of the L1 molecule, then inhibition of aggregation by ethanol should be observed in any cell type that expresses L1. Here we examined the effect of physiologically relevant concentrations of ethanol on the aggregation of Drosophila S2 cells that expressed either neuroglian (the Drosophila homolog of L1) or human L1. The aggregation of these S2 cells is known to be solely dependent on the homophilic interactions between L1 or neuroglian molecules. Neither cell adhesion molecule was affected when cell aggregation assays were carried out in the presence of >/=38 mM ethanol. The recruitment of membrane skeleton assembly at sites of cell-cell contact (a transmembrane signaling function of human L1) was also unaffected by the presence of ethanol. Thus the previously described inhibition of cell adhesion by ethanol in L1-expressing cells cannot be explained by a simple direct effect on the adhesive activity of L1 family members.

Influence of surface coating on the intracellular behaviour of gold nanoparticles: a fluorescence correlation spectroscopy study.

PubMed

Silvestri, A; Di Silvio, D; Llarena, I; Murray, R A; Marelli, M; Lay, L; Polito, L; Moya, S E

2017-10-05

In the biomedical applications of nanoparticles (NPs), the proper choice of surface chemistry is a crucial aspect in their design. The nature of the coating can heavily impact the interaction of NPs with biomolecules, affect the state of aggregation, and ultimately determine their biological fate. As such, protein corona formation and the aggregation behaviour of gold NPs (Au NPs) are studied here. Au NPs are prepared with four distinct surface functionalisations, namely mercaptosuccinic acid (MSA), N-4-thiobutyroil glucosamine, HS-PEG 5000 and HS-alkyl-PEG 600 . Corona formation, aggregation, and the intracellular behaviour of the Au NPs are then investigated by means of Fluorescence Correlation Spectroscopy (FCS) in cell culture media and in live cells. To evaluate the state of aggregation and the formation of a protein corona, the Au NPs are incubated in cell media and the diffusion coefficient is determined via FCS. The in vitro behaviour is compared with the level of aggregation of the NPs in cells. Diffusion times of the NPs are estimated at different positions in the cell after a one hour incubation period. It is found that the majority of MSA and glucose-Au NPs are present inside the cell as slowly diffusing species with diffusion times (τ D ) greater than 6000 μs (hydrodynamic diameter >250 nm). PEGylated Au NPs adsorb a small amount of protein and manifest low agglomeration both in media and in living cells. In particular, the HS-alkyl-PEG 600 coating shows an excellent correlation between lower protein adsorption, 4-fold lower compared to the MSA coated NPs, and limited intracellular aggregation. In the case of single HS-alkyl-PEG 600 coated NPs, it is found that typical intracellular τ D values range from 500 to 1500 μs, indicating that these particles display reduced aggregation in the intracellular environment.

Integration of Vibrio vulnificus into Marine Aggregates and Its Subsequent Uptake by Crassostrea virginica Oysters

PubMed Central

Froelich, Brett; Ayrapetyan, Mesrop

2013-01-01

Marine aggregates are naturally forming conglomerations of larvacean houses, phytoplankton, microbes, and inorganics adhered together by exocellular polymers. In this study, we show in vitro that the bacterial pathogen Vibrio vulnificus can be concentrated into laboratory-generated aggregates from surrounding water. We further show that environmental (E-genotype) strains exhibit significantly more integration into these aggregates than clinical (C-genotype) strains. Experiments where marine aggregates with attached V. vulnificus cells were fed to oysters (Crassostrea virginica) resulted in greater uptake of both C and E types than nonaggregated controls. When C- and E-genotype strains were cocultured in competitive experiments, the aggregated E-genotype strains exhibited significantly greater uptake by oyster than the C-genotype strains. PMID:23263962

Scanning electron microscopy study of adhesion in sea urchin blastulae. M.S. Thesis

NASA Technical Reports Server (NTRS)

Crowther, Susan D.

1988-01-01

The dissociation supernatant (DS) isolated by disaggregating Strongylocentrotus purpuratus blastulae in calcium- and magnesium-free seawater specifically promotes reaggregation of S. purpuratus blastula cells. The purpose of this study was to use scanning electron microscopy to examine the gross morphology of aggregates formed in the presence of DS to see if it resembles adhesion in partially dissociated blastulae. A new reaggregation procedure developed here, using large volumes of cell suspension and a large diameter of rotation, was utilized to obtain sufficient quantities of aggregates for scanning electron microscopy. The results indicate that aggregates formed in the presence of DS resemble partially dissociated intact embryos in terms of the direct cell-cell adhesion observed. DS did not cause aggregation to form as a result of the entrapment of cells in masses of extracellular material. These studies provide the groundwork for further studies using transmission electron microscopy to more precisely define the adhesive contacts made by cells in the presence of the putative adhesion molecules present in DS.

H-aggregate analysis of P3HT thin films-Capability and limitation of photoluminescence and UV/Vis spectroscopy.

PubMed

Ehrenreich, Philipp; Birkhold, Susanne T; Zimmermann, Eugen; Hu, Hao; Kim, Kwang-Dae; Weickert, Jonas; Pfadler, Thomas; Schmidt-Mende, Lukas

2016-09-01

Polymer morphology and aggregation play an essential role for efficient charge carrier transport and charge separation in polymer-based electronic devices. It is a common method to apply the H-aggregate model to UV/Vis or photoluminescence spectra in order to analyze polymer aggregation. In this work we present strategies to obtain reliable and conclusive information on polymer aggregation and morphology based on the application of an H-aggregate analysis on UV/Vis and photoluminescence spectra. We demonstrate, with P3HT as model system, that thickness dependent reflection behavior can lead to misinterpretation of UV/Vis spectra within the H-aggregate model. Values for the exciton bandwidth can deviate by a factor of two for polymer thicknesses below 150 nm. In contrast, photoluminescence spectra are found to be a reliable basis for characterization of polymer aggregation due to their weaker dependence on the wavelength dependent refractive index of the polymer. We demonstrate this by studying the influence of surface characteristics on polymer aggregation for spin-coated thin-films that are commonly used in organic and hybrid solar cells.

Kin discrimination increases with genetic distance in a social amoeba.

PubMed

Ostrowski, Elizabeth A; Katoh, Mariko; Shaulsky, Gad; Queller, David C; Strassmann, Joan E

2008-11-25

In the social amoeba Dictyostelium discoideum, thousands of cells aggregate upon starvation to form a multicellular fruiting body, and approximately 20% of them die to form a stalk that benefits the others. The aggregative nature of multicellular development makes the cells vulnerable to exploitation by cheaters, and the potential for cheating is indeed high. Cells might avoid being victimized if they can discriminate among individuals and avoid those that are genetically different. We tested how widely social amoebae cooperate by mixing isolates from different localities that cover most of their natural range. We show here that different isolates partially exclude one another during aggregation, and there is a positive relationship between the extent of this exclusion and the genetic distance between strains. Our findings demonstrate that D. discoideum cells co-aggregate more with genetically similar than dissimilar individuals, suggesting the existence of a mechanism that discerns the degree of genetic similarity between individuals in this social microorganism.

A mechanistic model for the evolution of multicellularity

NASA Astrophysics Data System (ADS)

Amado, André; Batista, Carlos; Campos, Paulo R. A.

2018-02-01

Through a mechanistic approach we investigate the formation of aggregates of variable sizes, accounting mechanisms of aggregation, dissociation, death and reproduction. In our model, cells can produce two metabolites, but the simultaneous production of both metabolites is costly in terms of fitness. Thus, the formation of larger groups can favor the aggregates to evolve to a configuration where division of labor arises. It is assumed that the states of the cells in a group are those that maximize organismal fitness. In the model it is considered that the groups can grow linearly, forming a chain, or compactly keeping a roughly spherical shape. Starting from a population consisting of single-celled organisms, we observe the formation of groups with variable sizes and usually much larger than two-cell aggregates. Natural selection can favor the formation of large groups, which allows the system to achieve new and larger fitness maxima.

Flower-shaped ZnO nanocrystallite aggregates synthesized through a template-free aqueous solution method for dye-sensitized solar cells

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

Chang, Wei-Chen, E-mail: changpeter@iner.gov.tw; Institute of Nuclear Energy Research, Atomic Energy Council, Executive Yuan, 1000 Wenhua Rd., Chiaan Village, Lungtan, Taoyuan 325, Taiwan; Chen, Hung-Shuo

Hierarchically structured flower-shaped aggregates composed of ZnO nanocrystals were synthesized through a template-free aqueous solution method. The synthesized nanocrystallite aggregates were demonstrated to be promising photoanode materials for dye-sensitized solar cells (DSSCs). Compared with commercially available ZnO nanoparticles (ZnONPs), the flower-like aggregates (ZnONFs), each having an overall dimension of 400–600 nm, exhibited similar dye loading but higher light-scattering ability, which led to a substantial increase in the light-harvesting efficiency of resulting cells. The unique morphology of ZnONFs also boosted the absorbed photon-to-electric current generation efficiency. Consequently, DSSCs constructed from ZnONFs showed significantly improved photocurrent and achieved an overall conversion efficiency ofmore » 4.42%, which was 47% higher than that attained by ZnONP-based cells.« less

Influence of thermal annealing-induced molecular aggregation on film properties and photovoltaic performance of bulk heterojunction solar cells based on a squaraine dye

NASA Astrophysics Data System (ADS)

Zhang, Pengpeng; Ling, Zhitian; Chen, Guo; Wei, Bin

2018-06-01

Squaraine (SQ) dyes have been considered as efficient photoactive materials for organic solar cells. In this work, we purposely controlled the molecular aggregation of an SQ dye, 2,4-bis[4-(N,N-dibutylamino)-2-dihydroxyphenyl] SQ (DBSQ-(OH)2) in the DBSQ(OH)2:[6,6]-phenyl-C61-butyric acid methyl ester (PCBM) blend film by using the thermal annealing method, to study the influence of the molecular aggregation on film properties as well as the photovoltaic performance of DBSQ(OH)2:PCBM-based bulk heterojunction (BHJ) solar cells. Our results demonstrate that thermal annealing may change the aggregation behavior of DBSQ(OH)2 in the DBSQ(OH)2:PCBM film, and thus significantly influence the surface morphology, optical and electrical properties of the blend film, as well as the photovoltaic performance of DBSQ(OH)2:PCBM BHJ cells.

Influence of thermal annealing-induced molecular aggregation on film properties and photovoltaic performance of bulk heterojunction solar cells based on a squaraine dye

NASA Astrophysics Data System (ADS)

Zhang, Pengpeng; Ling, Zhitian; Chen, Guo; Wei, Bin

2018-04-01

Squaraine (SQ) dyes have been considered as efficient photoactive materials for organic solar cells. In this work, we purposely controlled the molecular aggregation of an SQ dye, 2,4-bis[4-(N,N-dibutylamino)-2-dihydroxyphenyl] SQ (DBSQ-(OH)2) in the DBSQ(OH)2:[6,6]-phenyl-C61-butyric acid methyl ester (PCBM) blend film by using the thermal annealing method, to study the influence of the molecular aggregation on film properties as well as the photovoltaic performance of DBSQ(OH)2:PCBM-based bulk heterojunction (BHJ) solar cells. Our results demonstrate that thermal annealing may change the aggregation behavior of DBSQ(OH)2 in the DBSQ(OH)2:PCBM film, and thus significantly influence the surface morphology, optical and electrical properties of the blend film, as well as the photovoltaic performance of DBSQ(OH)2:PCBM BHJ cells.

Patterns of [PSI+] aggregation allow insights into cellular organization of yeast prion aggregates

PubMed Central

Tyedmers, Jens

2012-01-01

The yeast prion phenomenon is very widespread and mounting evidence suggests that it has an impact on cellular regulatory mechanisms related to phenotypic responses to changing environments. Studying the aggregation patterns of prion amyloids during different stages of the prion life cycle is a first key step to understand major principles of how and where cells generate, organize and turn-over prion aggregates. The induction of the [PSI+] state involves the actin cytoskeleton and quality control compartments such as the Insoluble Protein Deposit (IPOD). An initially unstable transitional induction state can be visualized by overexpression of the prion determinant and displays characteristic large ring- and ribbon-shaped aggregates consisting of poorly fragmented bundles of very long prion fibrils. In the mature prion state, the aggregation pattern is characterized by highly fragmented, shorter prion fibrils that form aggregates, which can be visualized through tagging with fluorescent proteins. The number of aggregates formed varies, ranging from a single large aggregate at the IPOD to multiple smaller ones, depending on several parameters discussed. Aggregate units below the resolution of light microscopy that are detectable by fluorescence correlation spectroscopy are in equilibrium with larger aggregates in this stage and can mediate faithful inheritance of the prion state. Loss of the prion state is often characterized by reduced fragmentation of prion fibrils and fewer, larger aggregates. PMID:22449721

Visualization of reticulophagy in living cells using an endoplasmic reticulum-targeted p62 mutant.

PubMed

Wang, Liang; Liu, Lei; Qin, Lingsong; Luo, Qingming; Zhang, Zhihong

2017-04-01

Reticulophagy is a type of selective autophagy in which protein aggregate-containing and/or damaged endoplasmic reticulum (ER) fragments are engulfed for lysosomal degradation, which is important for ER homeostasis. Several chemical drugs and mutant proteins that promote protein aggregate formation within the ER lumen can efficiently induce reticulophagy in mammalian cells. However, the exact mechanism and cellular localization of reticulophagy remain unclear. In this report, we took advantage of the self-oligomerization property of p62/SQSTM1, an adaptor for selective autophagy, and developed a novel reticulophagy system based on an ER-targeted p62 mutant to investigate the process of reticulophagy in living cells. LC3 conversion analysis via western blot suggested that p62 mutant aggregate-induced ER stress triggered a cellular autophagic response. Confocal imaging showed that in cells with moderate aggregation conditions, the aggregates of ER-targeted p62 mutants were efficiently sequestered by autophagosomes, which was characterized by colocalization with the autophagosome precursor marker ATG16L1, the omegasome marker DFCP1, and the late autophagosomal marker LC3/GATE-16. Moreover, time-lapse imaging data demonstrated that the LC3- or DFCP1-positive protein aggregates are tightly associated with the reticular structures of the ER, thereby suggesting that reticulophagy occurs at the ER and that omegasomes may be involved in this process.

Native aggregation as a cause of origin of temporary cellular structures needed for all forms of cellular activity, signaling and transformations.

PubMed

Matveev, Vladimir V

2010-06-09

According to the hypothesis explored in this paper, native aggregation is genetically controlled (programmed) reversible aggregation that occurs when interacting proteins form new temporary structures through highly specific interactions. It is assumed that Anfinsen's dogma may be extended to protein aggregation: composition and amino acid sequence determine not only the secondary and tertiary structure of single protein, but also the structure of protein aggregates (associates). Cell function is considered as a transition between two states (two states model), the resting state and state of activity (this applies to the cell as a whole and to its individual structures). In the resting state, the key proteins are found in the following inactive forms: natively unfolded and globular. When the cell is activated, secondary structures appear in natively unfolded proteins (including unfolded regions in other proteins), and globular proteins begin to melt and their secondary structures become available for interaction with the secondary structures of other proteins. These temporary secondary structures provide a means for highly specific interactions between proteins. As a result, native aggregation creates temporary structures necessary for cell activity."One of the principal objects of theoretical research in any department of knowledge is to find the point of view from which the subject appears in its greatest simplicity."Josiah Willard Gibbs (1839-1903).

Microfluidic-Based Measurement Method of Red Blood Cell Aggregation under Hematocrit Variations

PubMed Central

2017-01-01

Red blood cell (RBC) aggregation and erythrocyte sedimentation rate (ESR) are considered to be promising biomarkers for effectively monitoring blood rheology at extremely low shear rates. In this study, a microfluidic-based measurement technique is suggested to evaluate RBC aggregation under hematocrit variations due to the continuous ESR. After the pipette tip is tightly fitted into an inlet port, a disposable suction pump is connected to the outlet port through a polyethylene tube. After dropping blood (approximately 0.2 mL) into the pipette tip, the blood flow can be started and stopped by periodically operating a pinch valve. To evaluate variations in RBC aggregation due to the continuous ESR, an EAI (Erythrocyte-sedimentation-rate Aggregation Index) is newly suggested, which uses temporal variations of image intensity. To demonstrate the proposed method, the dynamic characterization of the disposable suction pump is first quantitatively measured by varying the hematocrit levels and cavity volume of the suction pump. Next, variations in RBC aggregation and ESR are quantified by varying the hematocrit levels. The conventional aggregation index (AI) is maintained constant, unrelated to the hematocrit values. However, the EAI significantly decreased with respect to the hematocrit values. Thus, the EAI is more effective than the AI for monitoring variations in RBC aggregation due to the ESR. Lastly, the proposed method is employed to detect aggregated blood and thermally-induced blood. The EAI gradually increased as the concentration of a dextran solution increased. In addition, the EAI significantly decreased for thermally-induced blood. From this experimental demonstration, the proposed method is able to effectively measure variations in RBC aggregation due to continuous hematocrit variations, especially by quantifying the EAI. PMID:28878199

1,2,4-trihydroxynaphthalene-2-O-β-D-glucopyranoside delays amyloid-β42 aggregation and reduces amyloid cytotoxicity.

PubMed

Dhouafli, Zohra; Leri, Manuela; Bucciantini, Monica; Stefani, Massimo; Gadhoumi, Hamza; Mahjoub, Borhane; Ben Jannet, Hichem; Guillard, Jérôme; Ksouri, Riadh; Saidani Tounsi, Moufida; Soto, Claudio; Hayouni, El Akrem

2018-03-26

Presently, misfolding and aggregation of amyloid-β 42 (Aβ 42 ) are considered early events in Alzheimer's disease (AD) pathogenesis. The use of natural products to inhibit the aggregation process and to protect cells from cytotoxicity of early aggregate grown at the onset of the aggregation path is one of the promising strategies against AD. Recently, we have purified a new powerful antioxidant and inhibitor of Aβ 42 aggregation from the leaves of Lawsonia inermis. The new compound was identified as a new Lawsoniaside; 1,2,4-trihydroxynaphthalene-2-O-β-D-glucopyranoside (THNG). Herein, we show that THNG interferes with Aβ 42 aggregation, inhibits its conformational change to a β-sheet-rich structure, decreases its polymerization into large fibrillar species, reduces oxidative stress, and aggregate cytotoxicity. These results indicate that THNG has great potential as a neuroprotective and therapeutic agent against AD. © 2018 BioFactors, 2018. © 2018 International Union of Biochemistry and Molecular Biology.

Dynamic hydrostatic pressure enhances differentially the chondrogenesis of meniscal cells from the inner and outer zone.

PubMed

Zellner, J; Mueller, M; Xin, Y; Krutsch, W; Brandl, A; Kujat, R; Nerlich, M; Angele, P

2015-06-01

This study analyses the influence of dynamic hydrostatic pressure on chondrogenesis of human meniscus-derived fibrochondrocytes and explores the differences in chondrogenic differentiation under loading conditions between cells derived from the avascular inner zone and vascularized outer region of the meniscus. Aggregates of human fibrochondrocytes with cell origin from the inner region or with cell origin from the outer region were generated. From the two groups of either cell origin, aggregates were treated with dynamic hydrostatic pressure (1Hz for 4h; 0.55-5.03MPa, cyclic sinusoidal) from day 1 to day 7. The other aggregates served as unloaded controls. At day 0, 7, 14 and 21 aggregates were harvested for evaluation including histology, immunostaining and ELISA analysis for glycosaminoglycan (GAG) and collagen II. Loaded aggregates were found to be macroscopically larger and revealed immunohistochemically enhanced chondrogenesis compared to the corresponding controls. Loaded or non-loaded meniscal cells from the outer zone showed a higher potential and earlier onset of chondrogenesis compared to the cells from the inner part of the meniscus. This study suggests that intrinsic factors like cell properties in the different areas of the meniscus and their reaction on mechanical load might play important roles in designing Tissue Engineering strategies for meniscal repair in vivo. Copyright © 2015 Elsevier Ltd. All rights reserved.

Early-Aggregation Studies of Polyglutamine in Solution

NASA Astrophysics Data System (ADS)

Fluitt, Aaron; de Pablo, Juan

2012-02-01

Several neurodegenerative diseases, notably Huntington's disease, are associated with certain proteins containing extended polyglutamine tracts. In all polyglutamine diseases, the age of onset is inversely correlated with the length of the polyglutamine domain beyond some pathological threshold. Diseased cells are characterized by intranuclear inclusions rich in aggregated polyglutamine. Experimental evidence suggests that oligomeric aggregate species, not mature amyloid fibrils, are the species most toxic to the cell. Little is known about the structures and aggregation dynamics of polyglutamine oligomers due to their short lifetimes. A better understanding of the pathway through which polyglutamine peptides form oligomeric aggregates will aid the design of therapies to inhibit their toxic activity. In this work, we report structural characterization of polyglutamine monomers and dimers from atomistic molecular dynamics simulations in explicit water. Umbrella sampling simulations reveal that the stability of the dimer species with respect to the disassociated monomers is an increasing function of the chain length.

Protein Misfolding Diseases.

PubMed

Hartl, F Ulrich

2017-06-20

The majority of protein molecules must fold into defined three-dimensional structures to acquire functional activity. However, protein chains can adopt a multitude of conformational states, and their biologically active conformation is often only marginally stable. Metastable proteins tend to populate misfolded species that are prone to forming toxic aggregates, including soluble oligomers and fibrillar amyloid deposits, which are linked with neurodegeneration in Alzheimer and Parkinson disease, and many other pathologies. To prevent or regulate protein aggregation, all cells contain an extensive protein homeostasis (or proteostasis) network comprising molecular chaperones and other factors. These defense systems tend to decline during aging, facilitating the manifestation of aggregate deposition diseases. This volume of the Annual Review of Biochemistry contains a set of three articles addressing our current understanding of the structures of pathological protein aggregates and their associated disease mechanisms. These articles also discuss recent insights into the strategies cells have evolved to neutralize toxic aggregates by sequestering them in specific cellular locations.

Sponge cell reaggregation: Cellular structure and morphogenetic potencies of multicellular aggregates.

PubMed

Lavrov, Andrey I; Kosevich, Igor A

2016-02-01

Sponges (phylum Porifera) are one of the most ancient extant multicellular animals and can provide valuable insights into origin and early evolution of Metazoa. High plasticity of cell differentiations and anatomical structure is characteristic feature of sponges. Present study deals with sponge cell reaggregation after dissociation as the most outstanding case of sponge plasticity. Dynamic of cell reaggregation and structure of multicellular aggregates of three demosponge species (Halichondria panicea (Pallas, 1766), Haliclona aquaeductus (Sсhmidt, 1862), and Halisarca dujardinii Johnston, 1842) were studied. Sponge tissue dissociation was performed mechanically. Resulting cell suspensions were cultured at 8-10°C for at least 5 days. Structure of multicellular aggregates was studied by light, transmission and scanning electron microscopy. Studied species share common stages of cell reaggregation-primary multicellular aggregates, early-stage primmorphs and primmorphs, but the rate of reaggregation varies considerably among species. Only cells of H. dujardinii are able to reconstruct functional and viable sponge after primmorphs formation. Sponge reconstruction in this species occurs due to active cell locomotion. Development of H. aquaeductus and H. panicea cells ceases at the stages of early primmorphs and primmorphs, respectively. Development of aggregates of these species is most likely arrested due to immobility of the majority of cells inside them. However, the inability of certain sponge species to reconstruct functional and viable individuals during cell reaggregation may be not a permanent species-specific characteristic, but depends on various factors, including the stage of the life cycle and experimental conditions. © 2016 Wiley Periodicals, Inc.

The cell aggregating propensity of probiotic actinobacterial isolates: isolation and characterization of the aggregation inducing peptide pheromone.

PubMed

Muthu Selvam, Ramu; Vinothini, Gopal; Palliyarai Thaiyammal, Sethuramalingam; Latha, Selvanathan; Chinnathambi, Arunachalam; Dhanasekaran, Dharumadurai; Padmanabhan, Parasuraman; Ali Alharbi, Sulaiman; Archunan, Govindaraju

2016-01-01

The auto-aggregating ability of a probiotic is a prerequisite for colonization and protection of the gastrointestinal tract, whereas co-aggregation provides a close interaction with pathogenic bacteria. Peptide pheromone mediated signaling has been studied in several systems. However, it has not yet been explored in prokaryotes, especially actinobacteria. Hence, in the present study, the diffusible aggregation promoting factor was purified from the culture supernatant of a potent actinobacterial probiont and characterized using 20 different actinobacterial cultures isolated from the gut region of chicken and goat. The results showed that the pheromone-like compound induces the aggregation propensity of treated isolates. The factor was found to be a heat stable, acidic pH resistant, low molecular weight peptide which enhances the biofilm forming ability of other actinobacterial isolates. The aggregation promoting factor represents a bacterial sex factor (pheromone) and its characterization confirms its usage in the probiotic formulation.

Structure-based inhibitors of tau aggregation

NASA Astrophysics Data System (ADS)

Seidler, P. M.; Boyer, D. R.; Rodriguez, J. A.; Sawaya, M. R.; Cascio, D.; Murray, K.; Gonen, T.; Eisenberg, D. S.

2018-02-01

Aggregated tau protein is associated with over 20 neurological disorders, which include Alzheimer's disease. Previous work has shown that tau's sequence segments VQIINK and VQIVYK drive its aggregation, but inhibitors based on the structure of the VQIVYK segment only partially inhibit full-length tau aggregation and are ineffective at inhibiting seeding by full-length fibrils. Here we show that the VQIINK segment is the more powerful driver of tau aggregation. Two structures of this segment determined by the cryo-electron microscopy method micro-electron diffraction explain its dominant influence on tau aggregation. Of practical significance, the structures lead to the design of inhibitors that not only inhibit tau aggregation but also inhibit the ability of exogenous full-length tau fibrils to seed intracellular tau in HEK293 biosensor cells into amyloid. We also raise the possibility that the two VQIINK structures represent amyloid polymorphs of tau that may account for a subset of prion-like strains of tau.

Red blood cells aggregability measurement of coagulating blood in extracorporeal circulation system with multiple-frequency electrical impedance spectroscopy.

PubMed

Li, Jianping; Sapkota, Achyut; Kikuchi, Daisuke; Sakota, Daisuke; Maruyama, Osamu; Takei, Masahiro

2018-07-30

Red blood cells (RBCs) aggregability A G of coagulating blood in extracorporeal circulation system has been investigated under the condition of pulsatile flow. Relaxation frequency f c from the multiple-frequency electrical impedance spectroscopy is utilized to obtain RBCs aggregability A G . Compared with other methods, the proposed multiple-frequency electrical impedance method is much easier to obtain non-invasive measurement with high speed and good penetrability performance in biology tissues. Experimental results show that, RBCs aggregability A G in coagulating blood falls down with the thrombus formation while that in non-coagulation blood almost keeps the same value, which has a great agreement with the activated clotting time (ACT) fibrinogen concertation (F bg ) tests. Modified Hanai formula is proposed to quantitatively analyze the influence of RBCs aggregation on multiple-frequency electrical impedance measurement. The reduction of RBCs aggregability A G is associated with blood coagulation reaction, which indicates the feasibility of the high speed, compact and cheap on-line thrombus measurement biosensors in extracorporeal circulation systems. Copyright © 2018 Elsevier B.V. All rights reserved.

Direct monitoring of erythrocytes aggregation under the effect of the low-intensity magnetic field by measuring light transmission at wavelength 800 nm

NASA Astrophysics Data System (ADS)

Elblbesy, Mohamed A.

2017-12-01

Interacting electromagnetic field with the living organisms and cells became of the great interest in the last decade. Erythrocytes are the most common types of the blood cells and have unique rheological, electrical, and magnetic properties. Aggregation is one of the important characteristics of the erythrocytes which has a great impact in some clinical cases. The present study introduces a simple method to monitor the effect of static magnetic field on erythrocytes aggregation using light transmission. Features were extracted from the time course curve of the light transmission through the whole blood under different intensities of the magnetic field. The findings of this research showed that static magnetic field could influence the size and the rate of erythrocytes aggregation. The strong correlations confirmed these results between the static magnetic field intensity and both the time of aggregation and sedimentation of erythrocytes. From this study, it can be concluded that static magnetic field can be used to modify the mechanisms of erythrocytes aggregation.

Protofibrils, pores, fibrils, and neurodegeneration: separating the responsible protein aggregates from the innocent bystanders.

PubMed

Caughey, Byron; Lansbury, Peter T

2003-01-01

Many neurodegenerative diseases, including Alzheimer's and Parkinson's and the transmissible spongiform encephalopathies (prion diseases), are characterized at autopsy by neuronal loss and protein aggregates that are typically fibrillar. A convergence of evidence strongly suggests that protein aggregation is neurotoxic and not a product of cell death. However, the identity of the neurotoxic aggregate and the mechanism by which it disables and eventually kills a neuron are unknown. Both biophysical studies aimed at elucidating the precise mechanism of in vitro aggregation and animal modeling studies support the emerging notion that an ordered prefibrillar oligomer, or protofibril, may be responsible for cell death and that the fibrillar form that is typically observed at autopsy may actually be neuroprotective. A subpopulation of protofibrils may function as pathogenic amyloid pores. An analogous mechanism may explain the neurotoxicity of the prion protein; recent data demonstrates that the disease-associated, infectious form of the prion protein differs from the neurotoxic species. This review focuses on recent experimental studies aimed at identification and characterization of the neurotoxic protein aggregates.

Identification of CLN6 as a molecular entity of endoplasmic reticulum-driven anti-aggregate activity.

PubMed

Yamashita, Arisa; Hiraki, Yuri; Yamazaki, Tetsuo

2017-06-10

αB-crystallin (αBC) is a small heat shock protein. Mutations in the αBC gene are linked to α-crystallinopathy, a hereditary myopathy histologically characterized by intracellular accumulation of protein aggregates. The disease-causing R120G αBC mutant, harboring an arginine-to-glycine replacement at position 120, is an aggregate-prone protein. We previously showed that the R120G mutant's aggregation in HeLa cells was prevented by enforced expression of αBC on the endoplasmic reticulum (ER). To elucidate the molecular nature of the preventive effect on the R120G mutant, we isolated proteins binding to ER-anchored αBC (TMαBC). The ER transmembrane CLN6 protein was identified as a TMαBC's binder. CLN6 knockdown in HeLa cells attenuated TMαBC's anti-aggregate activity against the R120G mutant. Conversely, CLN6 overexpression enhanced the activity, indicating that CLN6 operates as a downstream effector of TMαBC. CLN6 physically interacted with the R120G mutant, and repressed its aggregation in HeLa cells even when TMαBC was not co-expressed. Furthermore, CLN6's antagonizing effect on the R120G mutant was compromised upon treatment with a lysosomal inhibitor, suggesting CLN6 requires the intact autophagy-lysosome system to prevent the R120G mutant from aggregating. We hence conclude that CLN6 is not only a molecular entity of the anti-aggregate activity conferred by the ER manipulation using TMαBC, but also serves as a potential target of therapeutic interventions. Copyright © 2017 Elsevier Inc. All rights reserved.

SEROLOGICAL ANALYSES OF CELLULAR SLIME-MOLD DEVELOPMENT I.

PubMed Central

Sonneborn, D. R.; Sussman, M.; Levine, L.

1964-01-01

Sonneborn, D. R. (Brandeis University, Waltham, Mass.), M. Sussman, and L. Levine. Serological analysis of cellular slime-mold development. I. Changes in antigenic activity during cell aggregation. J. Bacteriol. 87:1321–1329. 1964.—During aggregation in Dictyostelium discoideum, the concentration of a single antigenic determinant increased markedly, starting from very low or undetectable levels. Subsequently, the determinant appeared to segregate preferentially into the stalks of terminal fruiting bodies. Sera containing the antibody specific for this determinant inhibited the aggregation of D. discoideum without disturbing cell viability. The properties of the antigen during fractionation are consistent with the supposition that it may be a protein associated with the cell membrane. The ability or inability of three species to coaggregate with D. discoideum was correlated with the presence or absence of the antigenic determinant in aggregates of these species. PMID:14188709

The Cleavage Effect of Mesenchymal Stem Cell and Its Derived Matrix Metalloproteinase‐2 on Extracellular α‐Synuclein Aggregates in Parkinsonian Models

PubMed Central

Oh, Se Hee; Kim, Ha Na; Park, Hyun Jung; Shin, Jin Young; Kim, Dong Yeol

2016-01-01

Abstract Ample evidence has suggested that extracellular α‐synuclein aggregates would play key roles in the pathogenesis and progression of Parkinsonian disorders (PDs). In the present study, we investigated whether mesenchymal stem cells (MSCs) and their derived soluble factors could exert neuroprotective effects via proteolysis of extracellular α‐synuclein. When preformed α‐synuclein aggregates were incubated with MSC‐conditioned medium, α‐synuclein aggregates were disassembled, and insoluble and oligomeric forms of α‐synuclein were markedly decreased, thus leading to a significant increase in neuronal viability. In an animal study, MSC or MSC‐conditioned medium treatment decreased the expression of α‐synuclein oligomers and the induction of pathogenic α‐synuclein with an attenuation of apoptotic cell death signaling. Furthermore, we identified that matrix metalloproteinase‐2 (MMP‐2), a soluble factor derived from MSCs, played an important role in the degradation of extracellular α‐synuclein. Our data demonstrated that MSCs and their derived MMP‐2 exert neuroprotective properties through proteolysis of aggregated α‐synuclein in PD‐related microenvironments. Stem Cells Translational Medicine 2017;6:949–961 PMID:28297586

Aggregation of ALS-linked FUS mutant sequesters RNA binding proteins and impairs RNA granules formation

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

Takanashi, Keisuke; Yamaguchi, Atsushi, E-mail: atsyama@restaff.chiba-u.jp

Highlights: • Aggregation of ALS-linked FUS mutant sequesters ALS-associated RNA-binding proteins (FUS wt, hnRNP A1, and hnRNP A2). • Aggregation of ALS-linked FUS mutant sequesters SMN1 in the detergent-insoluble fraction. • Aggregation of ALS-linked FUS mutant reduced the number of speckles in the nucleus. • Overproduced ALS-linked FUS mutant reduced the number of processing-bodies (PBs). - Abstract: Protein aggregate/inclusion is one of hallmarks for neurodegenerative disorders including amyotrophic lateral sclerosis (ALS). FUS/TLS, one of causative genes for familial ALS, encodes a multifunctional DNA/RNA binding protein predominantly localized in the nucleus. C-terminal mutations in FUS/TLS cause the retention and the inclusionmore » of FUS/TLS mutants in the cytoplasm. In the present study, we examined the effects of ALS-linked FUS mutants on ALS-associated RNA binding proteins and RNA granules. FUS C-terminal mutants were diffusely mislocalized in the cytoplasm as small granules in transiently transfected SH-SY5Y cells, whereas large aggregates were spontaneously formed in ∼10% of those cells. hnRNP A1, hnRNP A2, and SMN1 as well as FUS wild type were assembled into stress granules under stress conditions, and these were also recruited to FUS mutant-derived spontaneous aggregates in the cytoplasm. These aggregates stalled poly(A) mRNAs and sequestered SMN1 in the detergent insoluble fraction, which also reduced the number of nuclear oligo(dT)-positive foci (speckles) in FISH (fluorescence in situ hybridization) assay. In addition, the number of P-bodies was decreased in cells harboring cytoplasmic granules of FUS P525L. These findings raise the possibility that ALS-linked C-terminal FUS mutants could sequester a variety of RNA binding proteins and mRNAs in the cytoplasmic aggregates, which could disrupt various aspects of RNA equilibrium and biogenesis.« less

Investigation of microorganisms involved in biosynthesis of the kefir grain.

PubMed

Wang, Sheng-Yao; Chen, Kun-Nan; Lo, Yung-Ming; Chiang, Ming-Lun; Chen, Hsi-Chia; Liu, Je-Ruei; Chen, Ming-Ju

2012-12-01

The purpose of this study was to understand the significance of each microorganism in grain formation by evaluating their microbial aggregation and cell surface properties during co-aggregation of LAB and yeasts together with an investigation of biofilm formation. Non-grain forming strains from viili were also evaluated as a comparison. Results indicated that the kefir grain strains, Lactobacillus kefiranofaciens and Saccharomyces turicensis possess strong auto-aggregation ability and that Lactobacillus kefiri shows significant biofilm formation properties. Significant co-aggregation was noted when S. turicensis and kefir LAB strains (Lb. kefiranofaciens and Lb. kefiri) were co-cultured. Most of the tested LAB strains are hydrophilic and had a negative charge on their cell surface. Only the kefir LAB strains, Lb. kefiranofaciens HL1 and Lb. kefiri HL2, possessed very high hydrophobicity and had a positive cell surface charge at pH 4.2. In contrast, the LAB and yeasts in viili did not show any significant self-aggregation or biofilm formation. Based on the above results, we propose that grain formation begins with the self-aggregation of Lb. kefiranofaciens and S. turicensis to form small granules. At this point, the biofilm producer, Lb. kefiri, then begins to attach to the surface of granules and co-aggregates with other organisms and components in the milk to form the grains. On sub-culturing, more organisms attach to the grains resulting in grain growth. When investigated by scanning electron microscopy, it was found that short-chain lactobacilli such as Lb. kefiri occupy the surface, while long-chain lactobacilli such as Lb. kefiranofaciens have aggregated towards the center of the kefir grains. These findings agree with the above hypothesis on the formation of grains. Taken together, this study demonstrates the importance of cell surface properties together with fermentation conditions to the formation of grains in kefir. Copyright © 2012 Elsevier Ltd. All rights reserved.

Nuclear inclusion bodies of mutant and wild-type p53 in cancer: a hallmark of p53 inactivation and proteostasis remodelling by p53 aggregation.

PubMed

De Smet, Frederik; Saiz Rubio, Mirian; Hompes, Daphne; Naus, Evelyne; De Baets, Greet; Langenberg, Tobias; Hipp, Mark S; Houben, Bert; Claes, Filip; Charbonneau, Sarah; Delgado Blanco, Javier; Plaisance, Stephane; Ramkissoon, Shakti; Ramkissoon, Lori; Simons, Colinda; van den Brandt, Piet; Weijenberg, Matty; Van England, Manon; Lambrechts, Sandrina; Amant, Frederic; D'Hoore, André; Ligon, Keith L; Sagaert, Xavier; Schymkowitz, Joost; Rousseau, Frederic

2017-05-01

Although p53 protein aggregates have been observed in cancer cell lines and tumour tissue, their impact in cancer remains largely unknown. Here, we extensively screened for p53 aggregation phenotypes in tumour biopsies, and identified nuclear inclusion bodies (nIBs) of transcriptionally inactive mutant or wild-type p53 as the most frequent aggregation-like phenotype across six different cancer types. p53-positive nIBs co-stained with nuclear aggregation markers, and shared molecular hallmarks of nIBs commonly found in neurodegenerative disorders. In cell culture, tumour-associated stress was a strong inducer of p53 aggregation and nIB formation. This was most prominent for mutant p53, but could also be observed in wild-type p53 cell lines, for which nIB formation correlated with the loss of p53's transcriptional activity. Importantly, protein aggregation also fuelled the dysregulation of the proteostasis network in the tumour cell by inducing a hyperactivated, oncogenic heat-shock response, to which tumours are commonly addicted, and by overloading the proteasomal degradation system, an observation that was most pronounced for structurally destabilized mutant p53. Patients showing tumours with p53-positive nIBs suffered from a poor clinical outcome, similar to those with loss of p53 expression, and tumour biopsies showed a differential proteostatic expression profile associated with p53-positive nIBs. p53-positive nIBs therefore highlight a malignant state of the tumour that results from the interplay between (1) the functional inactivation of p53 through mutation and/or aggregation, and (2) microenvironmental stress, a combination that catalyses proteostatic dysregulation. This study highlights several unexpected clinical, biological and therapeutically unexplored parallels between cancer and neurodegeneration. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Regulator LdhR and d-Lactate Dehydrogenase LdhA of Burkholderia multivorans Play Roles in Carbon Overflow and in Planktonic Cellular Aggregate Formation.

PubMed

Silva, Inês N; Ramires, Marcelo J; Azevedo, Lisa A; Guerreiro, Ana R; Tavares, Andreia C; Becker, Jörg D; Moreira, Leonilde M

2017-10-01

LysR-type transcriptional regulators (LTTRs) are the most commonly found regulators in Burkholderia cepacia complex, comprising opportunistic pathogens causing chronic respiratory infections in cystic fibrosis (CF) patients. Despite LTTRs being global regulators of pathogenicity in several types of bacteria, few have been characterized in Burkholderia Here, we show that gene ldhR of B. multivorans encoding an LTTR is cotranscribed with ldhA encoding a d-lactate dehydrogenase and evaluate their implication in virulence traits such as exopolysaccharide (EPS) synthesis and biofilm formation. A comparison of the wild type (WT) and its isogenic Δ ldhR mutant grown in medium with 2% d-glucose revealed a negative impact on EPS biosynthesis and on cell viability in the presence of LdhR. The loss of viability in WT cells was caused by intracellular acidification as a consequence of the cumulative secretion of organic acids, including d-lactate, which was absent from the Δ ldhR mutant supernatant. Furthermore, LdhR is implicated in the formation of planktonic cellular aggregates. WT cell aggregates reached 1,000 μm in size after 24 h in liquid cultures, in contrast to Δ ldhR mutant aggregates that never grew more than 60 μm. The overexpression of d-lactate dehydrogenase LdhA in the Δ ldhR mutant partially restored the formed aggregate size, suggesting a role for fermentation inside aggregates. Similar results were obtained for surface-attached biofilms, with WT cells producing more biofilm. A systematic evaluation of planktonic aggregates in Burkholderia CF clinical isolates showed aggregates in 40 of 74. As CF patients' lung environments are microaerophilic and bacteria are found as free aggregates/biofilms, LdhR and LdhA might have central roles in adapting to this environment. IMPORTANCE Cystic fibrosis patients often suffer from chronic respiratory infections caused by several types of microorganisms. Among them are the Burkholderia cepacia complex bacteria, which cause progressive deterioration of lung function that, in some patients, might develop into fatal necrotizing pneumoniae with bacteremia, known as "cepacia syndrome." Burkholderia pathogenesis is multifactorial as they express several virulence factors, form biofilms, and are highly resistant to antimicrobial compounds, making their eradication from the CF patients' airways very difficult. As Burkholderia is commonly found in CF lungs in the form of cell aggregates and biofilms, the need to investigate the mechanisms of cellular aggregation is obvious. In this study, we demonstrate the importance of a d-lactate dehydrogenase and a regulator in regulating carbon overflow, cellular aggregates, and surface-attached biofilm formation. This not only enhances our understanding of Burkholderia pathogenesis but can also lead to the development of drugs against these proteins to circumvent biofilm formation. Copyright © 2017 American Society for Microbiology.

Rotary culture enhances pre-osteoblast aggregation and mineralization.

PubMed

Facer, S R; Zaharias, R S; Andracki, M E; Lafoon, J; Hunter, S K; Schneider, G B

2005-06-01

Three-dimensional environments have been shown to enhance cell aggregation and osteoblast differentiation. Thus, we hypothesized that three-dimensional (3D) growth environments would enhance the mineralization rate of human embryonic palatal mesenchymal (HEPM) pre-osteoblasts. The objective of this study was to investigate the potential use of rotary cell culture systems (RCCS) as a means to enhance the osteogenic potential of pre-osteoblast cells. HEPM cells were cultured in a RCCS to create 3D enviroments. Tissue culture plastic (2D) cultures served as our control. 3D environments promoted three-dimensional aggregate formations. Increased calcium and phosphorus deposition was significantly enhanced three- to 18-fold (P < 0.001) in 3D cultures as compared with 2D environments. 3D cultures mineralized in 1 wk as compared with the 2D cultures, which took 4 wks, a decrease in time of nearly 75%. In conclusion, our studies demonstrated that 3D environments enhanced osteoblast cell aggregation and mineralization.

Preventing α-synuclein aggregation: the role of the small heat-shock molecular chaperone proteins.

PubMed

Cox, Dezerae; Carver, John A; Ecroyd, Heath

2014-09-01

Protein homeostasis, or proteostasis, is the process of maintaining the conformational and functional integrity of the proteome. The failure of proteostasis can result in the accumulation of non-native proteins leading to their aggregation and deposition in cells and in tissues. The amyloid fibrillar aggregation of the protein α-synuclein into Lewy bodies and Lewy neuritis is associated with neurodegenerative diseases classified as α-synucleinopathies, which include Parkinson's disease and dementia with Lewy bodies. The small heat-shock proteins (sHsps) are molecular chaperones that are one of the cell's first lines of defence against protein aggregation. They act to stabilise partially folded protein intermediates, in an ATP-independent manner, to maintain cellular proteostasis under stress conditions. Thus, the sHsps appear ideally suited to protect against α-synuclein aggregation, yet these fail to do so in the context of the α-synucleinopathies. This review discusses how sHsps interact with α-synuclein to prevent its aggregation and, in doing so, highlights the multi-faceted nature of the mechanisms used by sHsps to prevent the fibrillar aggregation of proteins. It also examines what factors may contribute to α-synuclein escaping the sHsp chaperones in the context of the α-synucleinopathies. Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.

PIGN prevents protein aggregation in the endoplasmic reticulum independently of its function in the GPI synthesis.

PubMed

Ihara, Shinji; Nakayama, Sohei; Murakami, Yoshiko; Suzuki, Emiko; Asakawa, Masayo; Kinoshita, Taroh; Sawa, Hitoshi

2017-02-01

Quality control of proteins in the endoplasmic reticulum (ER) is essential for ensuring the integrity of secretory proteins before their release into the extracellular space. Secretory proteins that fail to pass quality control form aggregates. Here we show the PIGN-1/PIGN is required for quality control in Caenorhabditis elegans and in mammalian cells. In C. elegans pign-1 mutants, several proteins fail to be secreted and instead form abnormal aggregation. PIGN-knockout HEK293 cells also showed similar protein aggregation. Although PIGN-1/PIGN is responsible for glycosylphosphatidylinositol (GPI)-anchor biosynthesis in the ER, certain mutations in C. elegans pign-1 caused protein aggregation in the ER without affecting GPI-anchor biosynthesis. These results show that PIGN-1/PIGN has a conserved and non-canonical function to prevent deleterious protein aggregation in the ER independently of the GPI-anchor biosynthesis. PIGN is a causative gene for some human diseases including multiple congenital seizure-related syndrome (MCAHS1). Two pign-1 mutations created by CRISPR/Cas9 that correspond to MCAHS1 also cause protein aggregation in the ER, implying that the dysfunction of the PIGN non-canonical function might affect symptoms of MCAHS1 and potentially those of other diseases. © 2017. Published by The Company of Biologists Ltd.

Activated stress response pathways within multicellular aggregates utilize an autocrine component.

PubMed

Jack, Graham D; Cabrera, M Carla; Manning, Michael L; Slaughter, Stephen M; Potts, Malcolm; Helm, Richard F

2007-04-01

Multicellular aggregates (spheroids) of primary human foreskin fibroblasts (HFF-2) and a glioblastoma cell line (T98G) entered and exited from long term (2 weeks) metabolic arrest utilizing an autocrine response. Cytokine production (specifically IFN-gamma) activated a Gadd45alpha/p38 pathway that led to increased AP-1 (c-jun and ATF3) transcription factor levels, augmenting cytokine production in an autocrine fashion. Whereas HFF-2 aggregates were capable of surviving long term arrest and recovery during NF-kappaB inhibition independent of JNK activation, T98G aggregates were not. Such endogenous processes are not easily observed with adherent monolayer cell culturing systems, strongly suggesting that more emphasis needs to be placed on determining the operational signal transduction cascades within multicellular aggregates. Extracellular inputs such as spheroid formation, arrest, and regrowth as monolayers invoke intracellular signaling responses converging at the AP-1 transcription factor level. Variations in responses are both cell type and transformation state dependent and require an autocrine cytokine component. The data are discussed in relation to the wounding response and avascular tumor growth mechanisms.

Murine aggregation chimeras and wholemount imaging in airway stem cell biology.

PubMed

Rosewell, Ian R; Giangreco, Adam

2012-01-01

Local tissue stem cells are known to exist in mammalian lungs but their role in epithelial maintenance remains unclear. We therefore developed murine aggregation chimera and wholemount imaging techniques to assess the contribution of these cells to lung homeostasis and repair. In this chapter we provide further details regarding the generation of murine aggregation chimera mice and their subsequent use in wholemount lung imaging. We also describe methods related to the interpretation of this data that allows for quantitative assessment of airway stem cell activation versus quiescence. Using these techniques, it is possible to compare the growth and differentiation capacity of various lung epithelial cells in normal, repairing, and diseased states.

Chlorine, Chloramine, Chlorine Dioxide, and Ozone Susceptibility of Mycobacterium avium

PubMed Central

Taylor, Robert H.; Falkinham, Joseph O.; Norton, Cheryl D.; LeChevallier, Mark W.

2000-01-01

Environmental and patient isolates of Mycobacterium avium were resistant to chlorine, monochloramine, chlorine dioxide, and ozone. For chlorine, the product of the disinfectant concentration (in parts per million) and the time (in minutes) to 99.9% inactivation for five M. avium strains ranged from 51 to 204. Chlorine susceptibility of cells was the same in washed cultures containing aggregates and in reduced aggregate fractions lacking aggregates. Cells of the more slowly growing strains were more resistant to chlorine than were cells of the more rapidly growing strains. Water-grown cells were 10-fold more resistant than medium-grown cells. Disinfectant resistance may be one factor promoting the persistence of M. avium in drinking water. PMID:10742264

Ralstonia insidiosa induces cell aggregation by Listeria monocytogenes

USDA-ARS?s Scientific Manuscript database

Biofilm formation is an important strategy for foodborne bacterial pathogens to survive in stressful environments such as fresh produce processing facilities. Bacterial cell aggregation strongly promotes the initiation of microcolonies and the formation of biofilms on abiological surfaces. We previ...

Roles of CD34+ cells and ALK5 signaling in the reconstruction of seminiferous tubule-like structures in 3-D re-aggregate culture of dissociated cells from neonatal mouse testes.

PubMed

Abe, Shin-Ichi; Abe, Kazuko; Zhang, Jidong; Harada, Tomoaki; Mizumoto, Go; Oshikawa, Hiroki; Akiyama, Haruhiko; Shimamura, Kenji

2017-01-01

Tissue reconstruction in vitro can provide, if successful, a refined and simple system to analyze the underlying mechanisms that drive the morphogenesis and maintain the ordered structure. We have recently succeeded in reconstruction of seminiferous cord-like and tubule-like structures using 3-D re-aggregate culture of dissociated testicular cells. In testis formation, endothelial cells that migrated from mesonephroi to embryonic gonads have been shown to be critical for development of testis cords, but how endothelial cells contribute to testis cord formation remains unknown. To decipher the roles of endothelial and peritubular cells in the reconstruction of cord-like and tubule-like structures, we investigated the behavior of CD34+ endothelial and p75+ cells, and peritubular myoid cells (PTMCs) in 3-D re-aggregate cultures of testicular cells. The results showed that these 3 types of cells had the capacity of re-aggregation on their own and with each other, and of segregation into 3 layers in a re-aggregate, which were very similar to interstitial and peritubular tissues in vivo. Observation of behaviors of fluorescent Sertoli cells and other non-fluorescent types of cells using testes from Sox9-EGFP transgenic mice showed dynamic cell movement and segregation in re-aggregate cultures. Cultures of testicular cells deprived of interstitial and peritubular cells resulted in dysmorphic structures, but re-addition of them restored tubule-like structures. Purified CD34+ cells in culture differentiated into p75+ cells and PTMCs. These results indicate that CD34+ cells differentiate into p75+ cells, which then differentiate into PTMCs. TGFβ signaling inhibitors, SB431542 and ALK5i, disturbed the reconstruction of cord-like and tubule-like structures, and the latter compromised re-construction of interstitial-like and peritubular-like structures, as well as the proliferation of CD34+, p75+, PTMCs, and Sertoli cells, and their movement and differentiation. These results indicate that CD34+ cells and signaling through ALK5 play pivotal roles in the morphogenesis of interstitial-like, peritubular-like and cord-like structures.

The chaperonin CCT promotes the formation of fibrillar aggregates of γ-tubulin.

PubMed

Pouchucq, Luis; Lobos-Ruiz, Pablo; Araya, Gissela; Valpuesta, José María; Monasterio, Octavio

2018-04-01

The type II chaperonin CCT is involved in the prevention of the pathogenesis of numerous human misfolding disorders, as it sequesters misfolded proteins, blocks their aggregation and helps them to achieve their native state. In addition, it has been reported that CCT can prevent the toxicity of non-client amyloidogenic proteins by the induction of non-toxic aggregates, leading to new insight in chaperonin function as an aggregate remodeling factor. Here we add experimental evidence to this alternative mechanism by which CCT actively promotes the formation of conformationally different aggregates of γ-tubulin, a non-amyloidogenic CCT client protein, which are mediated by specific CCT-γ-tubulin interactions. The in vitro-induced aggregates were in some cases long fiber polymers, which compete with the amorphous aggregates. Direct injection of unfolded purified γ-tubulin into single-cell zebra fish embryos allowed us to relate this in vitro activity with the in vivo formation of intracellular aggregates. Injection of a CCT-binding deficient γ-tubulin mutant dramatically diminished the size of the intracellular aggregates, increasing the toxicity of the misfolded protein. These results point to CCT having a role in the remodeling of aggregates, constituting one of its many functions in cellular proteostasis. Copyright © 2018. Published by Elsevier B.V.

Transient shear viscosity of weakly aggregating polystyrene latex dispersions

NASA Astrophysics Data System (ADS)

de Rooij, R.; Potanin, A. A.; van den Ende, D.; Mellema, J.

1994-04-01

The transient behavior of the viscosity (stress growth) of a weakly aggregating polystyrene latex dispersion after a step from a high shear rate to a lower shear rate has been measured and modeled. Single particles cluster together into spherical fractal aggregates. The steady state size of these aggregates is determined by the shear stresses exerted on the latter by the flow field. The restructuring process taking place when going from a starting situation with monodisperse spherical aggregates to larger monodisperse spherical aggregates is described by the capture of primary fractal aggregates by growing aggregates until a new steady state is reached. It is assumed that the aggregation mechanism is diffusion limited. The model is valid if the radii of primary aggregates Rprim are much smaller than the radii of the growing aggregates. Fitting the model to experimental data at two volume fractions and a number of step sizes in shear rate yielded physically reasonable values of Rprim at fractal dimensions 2.1≤df≤2.2. The latter range is in good agreement with the range 2.0≤df≤2.3 obtained from steady shear results. The experimental data have also been fitted to a numerical solution of the diffusion equation for primary aggregates for a cell model with moving boundary, also yielding 2.1≤df≤2.2. The range for df found from both approaches agrees well with the range df≊2.1-2.2 determined from computer simulations on diffusion-limited aggregation including restructuring or thermal breakup after formation of bonds. Thus a simple model has been put forward which may capture the basic features of the aggregating model dispersion on a microstructural level and leads to physically acceptable parameter values.

Effects of environmental factors on MSP21-25 aggregation indicate the roles of hydrophobic and electrostatic interactions in the aggregation process.

PubMed

Zhang, Xuecheng; Dong, Yuanqiu; Yu, Jigang; Tu, Xiaoming

2014-01-01

Merozoite surface protein 2 (MSP2), one of the most abundant proteins on the merozoite surface of Plasmodium falciparum, is recognized to be important for the parasite's invasion into the host cell and is thus a promising malaria vaccine candidate. However, mediated mainly by its conserved N-terminal 25 residues (MSP21-25), MSP2 readily forms amyloid fibril-like aggregates under physiological conditions in vitro, which impairs its potential as a vaccine component. In addition, there is evidence that MSP2 exists in aggregated forms on the merozoite surface in vivo. To elucidate the aggregation mechanism of MSP21-25 and thereby understand the behavior of MSP2 in vivo and find ways to avoid the aggregation of relevant vaccine in vitro, we investigated the effects of agitation, pH, salts, 1-anilinonaphthalene-8-sulfonic acid (ANS), trimethylamine N-oxide dihydrate (TMAO), urea, and sub-micellar sodium dodecyl sulfate (SDS) on the aggregation kinetics of MSP21-25 using thioflavin T (ThT) fluorescence. The results showed that MSP21-25 aggregation was accelerated by agitation, while repressed by acidic pHs. The salts promoted the aggregation in an anion nature-dependent pattern. Hydrophobic surface-binding agent ANS and detergent urea repressed MSP21-25 aggregation, in contrast to hydrophobic interaction strengthener TMAO, which enhanced the aggregation. Notably, sub-micellar SDS, contrary to its micellar form, promoted MSP21-25 aggregation significantly. Our data indicated that hydrophobic interactions are the predominant driving force of the nucleation of MSP21-25 aggregation, while the elongation is controlled mainly by electrostatic interactions. A kinetic model of MSP21-25 aggregation and its implication were also discussed.

Effects of in vitro endochondral priming and pre-vascularisation of human MSC cellular aggregates in vivo.

PubMed

Freeman, Fiona E; Allen, Ashley B; Stevens, Hazel Y; Guldberg, Robert E; McNamara, Laoise M

2015-11-05

During endochondral ossification, both the production of a cartilage template and the subsequent vascularisation of that template are essential precursors to bone tissue formation. Recent studies have found the application of both chondrogenic and vascular priming of mesenchymal stem cells (MSCs) enhanced the mineralisation potential of MSCs in vitro whilst also allowing for immature vessel formation. However, the in vivo viability, vascularisation and mineralisation potential of MSC aggregates that have been pre-conditioned in vitro by a combination of chondrogenic and vascular priming, has yet to be established. In this study, we test the hypothesis that a tissue regeneration approach that incorporates both chondrogenic priming of MSCs, to first form a cartilage template, and subsequent pre-vascularisation of the cartilage constructs, by co-culture with human umbilical vein endothelial cells (HUVECs) in vitro, will improve vessel infiltration and thus mineral formation once implanted in vivo. Human MSCs were chondrogenically primed for 21 days, after which they were co-cultured with MSCs and HUVECs and cultured in endothelial growth medium for another 21 days. These aggregates were then implanted subcutaneously in nude rats for 4 weeks. We used a combination of bioluminescent imaging, microcomputed tomography, histology (Masson's trichrome and Alizarin Red) and immunohistochemistry (CD31, CD146, and α-smooth actin) to assess the vascularisation and mineralisation potential of these MSC aggregates in vivo. Pre-vascularised cartilaginous aggregates were found to have mature endogenous vessels (indicated by α-smooth muscle actin walls and erythrocytes) after 4 weeks subcutaneous implantation, and also viable human MSCs (detected by bioluminescent imaging) 21 days after subcutaneous implantation. In contrast, aggregates that were not pre-vascularised had no vessels within the aggregate interior and human MSCs did not remain viable beyond 14 days. Interestingly, the pre-vascularised cartilaginous aggregates were also the only group to have mineralised nodules within the cellular aggregates, whereas mineralisation occurred in the alginate surrounding the aggregates for all other groups. Taken together these results indicate that a combined chondrogenic priming and pre-vascularisation approach for in vitro culture of MSC aggregates shows enhanced vessel formation and increased mineralisation within the cellular aggregate when implanted subcutaneously in vivo.

Amyloidogenic Regions and Interaction Surfaces Overlap in Globular Proteins Related to Conformational Diseases

PubMed Central

Castillo, Virginia; Ventura, Salvador

2009-01-01

Protein aggregation underlies a wide range of human disorders. The polypeptides involved in these pathologies might be intrinsically unstructured or display a defined 3D-structure. Little is known about how globular proteins aggregate into toxic assemblies under physiological conditions, where they display an initially folded conformation. Protein aggregation is, however, always initiated by the establishment of anomalous protein-protein interactions. Therefore, in the present work, we have explored the extent to which protein interaction surfaces and aggregation-prone regions overlap in globular proteins associated with conformational diseases. Computational analysis of the native complexes formed by these proteins shows that aggregation-prone regions do frequently overlap with protein interfaces. The spatial coincidence of interaction sites and aggregating regions suggests that the formation of functional complexes and the aggregation of their individual subunits might compete in the cell. Accordingly, single mutations affecting complex interface or stability usually result in the formation of toxic aggregates. It is suggested that the stabilization of existing interfaces in multimeric proteins or the formation of new complexes in monomeric polypeptides might become effective strategies to prevent disease-linked aggregation of globular proteins. PMID:19696882

Novel Aggregation Properties of Candida albicans Secreted Aspartyl Proteinase Sap6 Mediate Virulence in Oral Candidiasis

PubMed Central

Kumar, Rohitashw; Saraswat, Darpan; Tati, Swetha

2015-01-01

Candida albicans, a commensal fungus of the oral microbiome, causes oral candidiasis in humans with localized or systemic immune deficiencies. Secreted aspartic proteinases (Saps) are a family of 10 related proteases and are virulence factors due to their proteolytic activity, as well as their roles in adherence and colonization of host tissues. We found that mice infected sublingually with C. albicans cells overexpressing Sap6 (SAP6 OE and a Δsap8 strain) had thicker fungal plaques and more severe oral infection, while infection with the Δsap6 strain was attenuated. These hypervirulent strains had highly aggregative colony structure in vitro and higher secreted proteinase activity; however, the levels of proteinase activity of C. albicans Saps did not uniformly match their abilities to damage cultured oral epithelial cells (SCC-15 cells). Hyphal induction in cells overexpressing Sap6 (SAP6 OE and Δsap8 cells) resulted in formation of large cell-cell aggregates. These aggregates could be produced in germinated wild-type cells by addition of native or heat-inactivated Sap6. Sap6 bound only to germinated cells and increased C. albicans adhesion to oral epithelial cells. The adhesion properties of Sap6 were lost upon deletion of its integrin-binding motif (RGD) and could be inhibited by addition of RGD peptide or anti-integrin antibodies. Thus, Sap6 (but not Sap5) has an alternative novel function in cell-cell aggregation, independent of its proteinase activity, to promote infection and virulence in oral candidiasis. PMID:25870228

Novel Aggregation Properties of Candida albicans Secreted Aspartyl Proteinase Sap6 Mediate Virulence in Oral Candidiasis.

PubMed

Kumar, Rohitashw; Saraswat, Darpan; Tati, Swetha; Edgerton, Mira

2015-07-01

Candida albicans, a commensal fungus of the oral microbiome, causes oral candidiasis in humans with localized or systemic immune deficiencies. Secreted aspartic proteinases (Saps) are a family of 10 related proteases and are virulence factors due to their proteolytic activity, as well as their roles in adherence and colonization of host tissues. We found that mice infected sublingually with C. albicans cells overexpressing Sap6 (SAP6 OE and a Δsap8 strain) had thicker fungal plaques and more severe oral infection, while infection with the Δsap6 strain was attenuated. These hypervirulent strains had highly aggregative colony structure in vitro and higher secreted proteinase activity; however, the levels of proteinase activity of C. albicans Saps did not uniformly match their abilities to damage cultured oral epithelial cells (SCC-15 cells). Hyphal induction in cells overexpressing Sap6 (SAP6 OE and Δsap8 cells) resulted in formation of large cell-cell aggregates. These aggregates could be produced in germinated wild-type cells by addition of native or heat-inactivated Sap6. Sap6 bound only to germinated cells and increased C. albicans adhesion to oral epithelial cells. The adhesion properties of Sap6 were lost upon deletion of its integrin-binding motif (RGD) and could be inhibited by addition of RGD peptide or anti-integrin antibodies. Thus, Sap6 (but not Sap5) has an alternative novel function in cell-cell aggregation, independent of its proteinase activity, to promote infection and virulence in oral candidiasis.

Down regulation of ITGA4 and ITGA5 genes after formation of 3D spherules by human Wharton's jelly stem cells (hWJSCs).

PubMed

Mostafavi-Pour, Zohreh; Ashrafi, Mohammad Reza; Talaei-Khozani, Tahereh

2018-06-01

Human Wharton's jelly mesenchymal stem cells (hWJSCs) are multipotent stem cells that could be aggregated into 3D spherules. ITGA4 and ITGA5 genes encode α4 and α5 subunits of integrins, respectively. In this study, we analyzed expression levels of ITGA4 and ITGA5 gene mRNAs in undifferentiated and 3D spherules forming hWJSCs in order to determine their expression pattern for possible future treatment of cancer cells in a co-culture fashion. For the purpose of obtaining hWJSCs, umbilical cords were collected from patients with caesarian section at full term delivery. The cells were then characterized according to cell surface markers using flow cytometry. Furthermore pluripotency of the obtained cells was verified. Subsequently the cells were aggregated in 3D spherules using hanging drop cultures. Expression levels of ITGA4 and ITGA5 gene mRNAs were determined by RT-PCR and Real time PCR, both in the initial undifferentiated cells and those aggregated in the spherules. The obtained hWJSCs demonstrated pluripotency, differentiating to adipogenic and osteogenic cells. They also expressed mesenchymal stem cell surface markers. Following the aggregation of these cells and formation of 3D spherules, mRNA expression levels of both genes were significantly reduced (P < 0.05) compared with the initial undifferentiated state. The results of this study demonstrated that aggregation of hWJSCs into spherules alters their expression of ITGA4 and ITGA5. The implications of such an alteration would require further research.

A 3D Culture Model to Study How Fluid Pressure and Flow Affect the Behavior of Aggregates of Epithelial Cells.

PubMed

Piotrowski-Daspit, Alexandra S; Simi, Allison K; Pang, Mei-Fong; Tien, Joe; Nelson, Celeste M

2017-01-01

Cells are surrounded by mechanical stimuli in their microenvironment. It is important to determine how cells respond to the mechanical information that surrounds them in order to understand both development and disease progression, as well as to be able to predict cell behavior in response to physical stimuli. Here we describe a protocol to determine the effects of interstitial fluid flow on the migratory behavior of an aggregate of epithelial cells in a three-dimensional (3D) culture model. This protocol includes detailed methods for the fabrication of a 3D cell culture chamber with hydrostatic pressure control, the culture of epithelial cells as an aggregate in a collagen gel, and the analysis of collective cell behavior in response to pressure-driven flow.

Influence of Calcium in Extracellular DNA Mediated Bacterial Aggregation and Biofilm Formation

PubMed Central

Koop, Leena; Wong, Yie Kuan; Ahmed, Safia; Siddiqui, Khawar Sohail; Manefield, Mike

2014-01-01

Calcium (Ca2+) has an important structural role in guaranteeing the integrity of the outer lipopolysaccharide layer and cell walls of bacterial cells. Extracellular DNA (eDNA) being part of the slimy matrix produced by bacteria promotes biofilm formation through enhanced structural integrity of the matrix. Here, the concurrent role of Ca2+ and eDNA in mediating bacterial aggregation and biofilm formation was studied for the first time using a variety of bacterial strains and the thermodynamics of DNA to Ca2+ binding. It was found that the eDNA concentrations under both planktonic and biofilm growth conditions were different among bacterial strains. Whilst Ca2+ had no influence on eDNA release, presence of eDNA by itself favours bacterial aggregation via attractive acid-base interactions in addition, its binding with Ca2+ at biologically relevant concentrations was shown further increase in bacterial aggregation via cationic bridging. Negative Gibbs free energy (ΔG) values in iTC data confirmed that the interaction between DNA and Ca2+ is thermodynamically favourable and that the binding process is spontaneous and exothermic owing to its highly negative enthalpy. Removal of eDNA through DNase I treatment revealed that Ca2+ alone did not enhance cell aggregation and biofilm formation. This discovery signifies the importance of eDNA and concludes that existence of eDNA on bacterial cell surfaces is a key facilitator in binding of Ca2+ to eDNA thereby mediating bacterial aggregation and biofilm formation. PMID:24651318

Experiment on ``discovery'' STS 51-C: Aggregation of red cells and thrombocytes in heart disease, hyperlipidaemia and other conditions

NASA Astrophysics Data System (ADS)

Dintenfass, L.

The aim of this experiment was to study aggregation of red cells in the blood of patients with ischaemic heart disease, diabetes, hyperlipidaemia, and (silent) cancer, and in two normal donors. Reconstituted blood using IgG was also used. The instrument, the automated slit-capillary photo-viscometer (100 kg weight) was set on the middeck of the Space Shuttle. An analogous instrument was at the Kennedy Space Center. Blood was obtained from donors, anticoagulated, and adjusted to haematocrit of 30% using native plasma. Experiments took place at 25°C, during which blood was forced to flow in the slit formed by two parallel glass plates. Macro and microphotography was carried out at specific intervals controlled by a computer. During stasis, lasting 6 minutes, aggregates (or clumps) of the red cells were formed. Results indicated that red cell aggregates do form under zero-G; that such aggregates are smaller than the ones obtained at one-G; that morphology is different, the zero-G showing rouleaux while one-G showing usual sludge-like clumps of red cells in all severe disorders. Platelets appeared to remain monodisperse under zero-G. Assuming that these data can be confirmed, one could suggest that zero-G affects cell-cell interaction, and may consequently influence the internal microstructure of the cell membrane and of the receptors, as well as their activity. Gravitational studies may thus open a new door on immunology and haematology in general.

Rapid Aggregation of Biofuel-Producing Algae by the Bacterium Bacillus sp. Strain RP1137

PubMed Central

Powell, Ryan J.

2013-01-01

Algal biofuels represent one of the most promising means of sustainably replacing liquid fuels. However, significant challenges remain before alga-based fuels become competitive with fossil fuels. One of the largest challenges is the ability to harvest the algae in an economical and low-energy manner. In this article, we describe the isolation of a bacterial strain, Bacillus sp. strain RP1137, which can rapidly aggregate several algae that are candidates for biofuel production, including a Nannochloropsis sp. This bacterium aggregates algae in a pH-dependent and reversible manner and retains its aggregation ability after paraformaldehyde fixation, opening the possibility for reuse of the cells. The optimal ratio of bacteria to algae is described, as is the robustness of aggregation at different salinities and temperatures. Aggregation is dependent on the presence of calcium or magnesium ions. The efficiency of aggregation of Nannochloropsis oceanica IMET1 is between 70 and 95% and is comparable to that obtained by other means of harvest; however, the rate of harvest is fast, with aggregates forming in 30 s. PMID:23892750

[Platelet function in acute myeloid leukemia. II. Aggregation of isolated platelets].

PubMed

Zawilska, K; Komarnicki, M; Mańka, B

1978-01-01

In 22 patients with acute myeloid leukaemia (17 cases of myeloblastic leukaemia, 4 cases of myelomonocytic leukaemia and 1 case of undifferentiated-cell leukaemia) platelets were isolated from the plasma by the method of Nicholls and Hampton as modified by Levy-Toledano by centrifugation in albumin gradient. The aim of platelet isolation was their "concentration" in cases of thrombocytopenia to values making possible aggregation tests, and platelet separation from the influence of plasma factors. Then aggregation of isolated platelets caused by ADP was studied. In 16 out of 22 patients a fall of aggregation was observed, with the mean values of aggregation rate and intensity were significantly lower. Parallelly done determinations of aggregating activity released from the platelets by thrombin showed lower values as compared with platelets from healthy subjects. In might be thought, in this connection, that the demonstrated reduction of isolated platelets is associated with a diminution of the nucleotide pool or disturbances of the platelet release reaction. The disturbances of the platelet release reaction. The disturbances of aggregation of isolated platelets and reduction of the aggregating activity were most pronounced in acute myelomonocytic leukaemia.

Protein aggregates as depots for the release of biologically active compounds.

PubMed

Artemova, Natalya V; Kasakov, Alexei S; Bumagina, Zoya M; Lyutova, Elena M; Gurvits, Bella Ya

2008-12-12

Protein misfolding and aggregation is one of the most serious problems in cell biology, molecular medicine, and biotechnology. Misfolded proteins interact with each other or with other proteins in non-productive or damaging ways. However, a new paradigm arises that protein aggregation may be exploited by nature to perform specific functions in different biological contexts. From this consideration, acceleration of stress-induced protein aggregation triggered by any factor resulting in the formation of soluble aggregates may have paradoxical positive consequences. Here, we suggest that amorphous aggregates can act as a source for the release of biologically active proteins after removal of stress conditions. To address this concept, we investigated the kinetics of thermal aggregation in vitro of yeast alcohol dehydrogenase (ADH) as a model substrate in the presence of two amphiphilic peptides: Arg-Phe or Ala-Phe-Lys. Using dynamic light scattering (DLS) and turbidimetry, we have demonstrated that under mild stress conditions the concentration-dependent acceleration of ADH aggregation by these peptides results in formation of large but soluble complexes of proteins prone to refolding.

Physiological studies of chloramine resistance developed by Klebsiella pneumoniae under low-nutrient growth conditions.

PubMed Central

Stewart, M H; Olson, B H

1992-01-01

This study investigated the physiological mechanisms of resistance to chloramines developed by Klebsiella pneumoniae grown in a nutrient-limited environment. Growth under these conditions resulted in cells that were smaller than cells grown under high-nutrient conditions and extensively aggregated. Cellular aggregates ranged from 10 to more than 10,000 cells per aggregate, with a mean population aggregate size of 90 cells. This aggregation may have been facilitated by the presence of extracellular polymer material. By using glucose as a reference of capsule content, it was determined that growth under low-nutrient conditions produced cells with 8 x 10(-14) to 41 x 10(-14) g of carbohydrate per cell, with a mean +/- standard deviation of 27 x 10(-14) +/- 16 x 10(-14) g of carbohydrate per cell. In comparison, growth under high-nutrient conditions resulted in 2.7 x 10(-14) to 5.9 x 10(-14) g of carbohydrate per cell, with a mean and standard deviation of 4.3 x 10(-14) +/- 1.2 x 10(-14) g of carbohydrate per cell. Cell wall and cell membrane lipids also varied with growth conditions. The ratio of saturated to unsaturated fatty acids in cells grown under low-nutrient conditions was approximately five times greater than that in cells grown under high-nutrient conditions, suggesting possible differences in membrane permeability. An analysis of sulfhydryl (-SH) groups revealed no quantitative difference with respect to growth conditions. However, upon exposure to chloramines, only 33% of the -SH groups of cells grown under low-nutrient conditions were oxidized, compared with 80% oxidization of -SH groups in cells grown under high-nutrient conditions. The reduced effectiveness of chloramine oxidization of -SH groups in cells grown under low-nutrient conditions may be due to restricted penetration of chloramines into the cells, conformational changes of enzymes, or a combination of both factors. The results of this study suggest that chloramine resistance developed under low-nutrient growth conditions may be a function of multiple physiological factors, including cellular aggregation and protection of sulfhydryl groups within the cell. PMID:1444406

Stoichiometry of Nck-dependent actin polymerization in living cells

PubMed Central

Ditlev, Jonathon A.; Michalski, Paul J.; Huber, Greg; Rivera, Gonzalo M.; Mohler, William A.

2012-01-01

Regulation of actin dynamics through the Nck/N-WASp (neural Wiskott–Aldrich syndrome protein)/Arp2/3 pathway is essential for organogenesis, cell invasiveness, and pathogen infection. Although many of the proteins involved in this pathway are known, the detailed mechanism by which it functions remains undetermined. To examine the signaling mechanism, we used a two-pronged strategy involving computational modeling and quantitative experimentation. We developed predictions for Nck-dependent actin polymerization using the Virtual Cell software system. In addition, we used antibody-induced aggregation of membrane-targeted Nck SH3 domains to test these predictions and to determine how the number of molecules in Nck aggregates and the density of aggregates affected localized actin polymerization in living cells. Our results indicate that the density of Nck molecules in aggregates is a critical determinant of actin polymerization. Furthermore, results from both computational simulations and experimentation support a model in which the Nck/N-WASp/Arp2/3 stoichiometry is 4:2:1. These results provide new insight into activities involving localized actin polymerization, including tumor cell invasion, microbial pathogenesis, and T cell activation. PMID:22613834

Receptor mechanisms in fish chromatophores--VII. Muscarinic cholinoceptors and alpha adrenoceptors, both mediating pigment aggregation, strangely coexist in Corydoras melanophores.

PubMed

Kasukawa, H; Fujii, R

1985-01-01

Both acetylcholine and catecholamines showed melanin-aggregating action within melanophores on an isolated bony plate of the mailed catfish Corydoras paleatus. Chromatic nervous stimulation either by an electrical field or by an elevation of [K+]0 brought about melanosome aggregation. Alpha adrenolytic agents antagonized the melanin-aggregating effects either of catecholamines or of nervous stimuli. Muscarinic cholinolytics interfered with the action of acetylcholine, but did not have any effect on the responses to nervous stimuli. In addition to the alpha adrenoceptors which participate in sympathetic-melanophore transmission, muscarinic cholinoceptors of unknown functional significance, which also mediate melanosome aggregation in the cell, exist in Corydoras melanophores.

Tunneling nanotubes spread fibrillar α-synuclein by intercellular trafficking of lysosomes.

PubMed

Abounit, Saïda; Bousset, Luc; Loria, Frida; Zhu, Seng; de Chaumont, Fabrice; Pieri, Laura; Olivo-Marin, Jean-Christophe; Melki, Ronald; Zurzolo, Chiara

2016-10-04

Synucleinopathies such as Parkinson's disease are characterized by the pathological deposition of misfolded α-synuclein aggregates into inclusions throughout the central and peripheral nervous system. Mounting evidence suggests that intercellular propagation of α-synuclein aggregates may contribute to the neuropathology; however, the mechanism by which spread occurs is not fully understood. By using quantitative fluorescence microscopy with co-cultured neurons, here we show that α-synuclein fibrils efficiently transfer from donor to acceptor cells through tunneling nanotubes (TNTs) inside lysosomal vesicles. Following transfer through TNTs, α-synuclein fibrils are able to seed soluble α-synuclein aggregation in the cytosol of acceptor cells. We propose that donor cells overloaded with α-synuclein aggregates in lysosomes dispose of this material by hijacking TNT-mediated intercellular trafficking. Our findings thus reveal a possible novel role of TNTs and lysosomes in the progression of synucleinopathies. © 2016 The Authors.

Experiment on aggregation of red cells under microgravity on STS 51-C

NASA Astrophysics Data System (ADS)

Dintenfass, L.; Osman, P.; Maguire, B.; Jedrzejczyk, H.

Kinetics and morphology of aggregation of red cells were studied using automatic slit-capillary photo-viscometers, one situated on the middeck of the space shuttle `Discovery', and the other in the ground laboratory at KSC. Experiments were run simultaneously, blood samples being adjusted to haematocrit of 0.30 using native plasma, at temp. of 25°C, and anticoagulated by EDTA. Donors included patients with myocardial infarction, insulin-dependent diabetes, hyperlipidaemia and hypertension. Macro and microphotographs were obtained during flow and statis. There was a striking difference in the morphology of aggregates formed in space and on the ground. Aggregates formed under zero gravity showed rouleaux formation, while the same blood samples showed severe clumping on the ground, in all patients blood. Normal blood showed rouleaux on the ground, but a random swarm-like pattern in space. The shape of the red cells remained normal under zero gravity.

The investigation of interspecies diversity of erythrocyte aggregation properties by two different photometric methods in four animal species.

PubMed

Kiss, F; Toth, E; Peto, K; Miko, I; Nemeth, N

2015-12-01

Among the haemorheological parameters, red blood cell (RBC) aggregation shows the largest interspecies diversity, and often controversial data can be found in the literature, besides the methodology-dependent issues. In this present investigation, we compared four experimental/laboratory animal species' RBC aggregation by two different photometric methods for better revealing the differences. Blood samples (K3-EDTA, 1.5 mg/ml) were taken from female animals: 16 inbred mice (Mus musculus, cardiac puncture), 15 outbred rats (Rattus norvegicus, caudal caval vein puncture), 15 beagle dogs (Canis canis, cephalic vein) and 23 juvenile pigs (Sus scrofa domesticus, medial saphenous vein). Haematological parameters (microcell counter) and RBC aggregation (light transmission and syllectometry-laser backscatter methods) were determined within 2 h after sampling. Describing the first 5-10 s of the aggregation process, additional parameters were calculated out of the syllectometric raw data. Standardized difference was calculated to determine the sensitivity of the two devices. Parameters describing the extent and magnitude of red blood cell aggregation showed the lowest values in the rat and the highest in the pig and canine blood. In turn, parameters describing the kinetics of aggregation showed the lowest values in the mouse and the highest in the rat. The standardized difference values for the laser backscattering method were 2-4 times larger vs. the light transmission one. The magnitude of the differences was not consequent in the aggregation parameters. These comparative results show that the laser backscattering method can detect the RBC aggregation differences between the investigated species more sensitively than the light transmission method. Journal of Animal Physiology and Animal Nutrition © 2015 Blackwell Verlag GmbH.

Wetting of nonconserved residue-backbones: A feature indicative of aggregation associated regions of proteins.

PubMed

Pradhan, Mohan R; Pal, Arumay; Hu, Zhongqiao; Kannan, Srinivasaraghavan; Chee Keong, Kwoh; Lane, David P; Verma, Chandra S

2016-02-01

Aggregation is an irreversible form of protein complexation and often toxic to cells. The process entails partial or major unfolding that is largely driven by hydration. We model the role of hydration in aggregation using "Dehydrons." "Dehydrons" are unsatisfied backbone hydrogen bonds in proteins that seek shielding from water molecules by associating with ligands or proteins. We find that the residues at aggregation interfaces have hydrated backbones, and in contrast to other forms of protein-protein interactions, are under less evolutionary pressure to be conserved. Combining evolutionary conservation of residues and extent of backbone hydration allows us to distinguish regions on proteins associated with aggregation (non-conserved dehydron-residues) from other interaction interfaces (conserved dehydron-residues). This novel feature can complement the existing strategies used to investigate protein aggregation/complexation. © 2015 Wiley Periodicals, Inc.

Cell and Particle Interactions and Aggregation During Electrophoretic Motion

NASA Technical Reports Server (NTRS)

Davis, Robert H.

2000-01-01

The objectives of this research were (i) to perform experiments for observing and quantifying electrophoretic aggregation, (ii) to develop a theoretical description to appropriately analyze and compare with the experimental results, (iii) to study the combined effects of electrophoretic and gravitational aggregation of large particles, and the combined effects of electrophoretic and Brownian aggregation of small particles, and (iv) to perform a preliminary design of a potential future flight experiment involving electrophoretic aggregation. Electrophoresis refers to the motion of charged particles, droplets or molecules in response to an applied electric field. Electrophoresis is commonly used for analysis and separation of biological particles or molecules. When particles have different surface charge densities or potentials, they will migrate at different velocities in an electric field. This differential migration leads to the possibility that they will collide and aggregate, thereby preventing separation.

A mechanistic model of tau amyloid aggregation based on direct observation of oligomers

NASA Astrophysics Data System (ADS)

Shammas, Sarah L.; Garcia, Gonzalo A.; Kumar, Satish; Kjaergaard, Magnus; Horrocks, Mathew H.; Shivji, Nadia; Mandelkow, Eva; Knowles, Tuomas P. J.; Mandelkow, Eckhard; Klenerman, David

2015-04-01

Protein aggregation plays a key role in neurodegenerative disease, giving rise to small oligomers that may become cytotoxic to cells. The fundamental microscopic reactions taking place during aggregation, and their rate constants, have been difficult to determine due to lack of suitable methods to identify and follow the low concentration of oligomers over time. Here we use single-molecule fluorescence to study the aggregation of the repeat domain of tau (K18), and two mutant forms linked with familial frontotemporal dementia, the deletion mutant ΔK280 and the point mutant P301L. Our kinetic analysis reveals that aggregation proceeds via monomeric assembly into small oligomers, and a subsequent slow structural conversion step before fibril formation. Using this approach, we have been able to quantitatively determine how these mutations alter the aggregation energy landscape.

Hypertonic stress induces rapid and widespread protein damage in C. elegans

PubMed Central

Burkewitz, Kris; Choe, Keith

2011-01-01

Proteostasis is defined as the homeostatic mechanisms that maintain the function of all cytoplasmic proteins. We recently demonstrated that the capacity of the proteostasis network is a critical factor that defines the limits of cellular and organismal survival in hypertonic environments. The current studies were performed to determine the extent of protein damage induced by cellular water loss. Using worm strains expressing fluorescently tagged foreign and endogenous proteins and proteins with temperature-sensitive point mutations, we demonstrate that hypertonic stress causes aggregation and misfolding of diverse proteins in multiple cell types. Protein damage is rapid. Aggregation of a polyglutamine yellow fluorescent protein reporter is observable with <1 h of hypertonic stress, and aggregate volume doubles approximately every 10 min. Aggregate formation is irreversible and occurs after as little as 10 min of exposure to hypertonic conditions. To determine whether endogenous proteins are aggregated by hypertonic stress, we quantified the relative amount of total cellular protein present in detergent-insoluble extracts. Exposure for 4 h to 400 mM or 500 mM NaCl induced a 55–120% increase in endogenous protein aggregation. Inhibition of insulin signaling or acclimation to mild hypertonic stress increased survival under extreme hypertonic conditions and prevented aggregation of endogenous proteins. Our results demonstrate that hypertonic stress causes widespread and dramatic protein damage and that cells have a significant capacity to remodel the network of proteins that function to maintain proteostasis. These findings have important implications for understanding how cells cope with hypertonic stress and other protein-damaging stressors. PMID:21613604

Effects of red blood cell aggregates dissociation on the estimation of ultrasound speckle image velocimetry.

PubMed

Yeom, Eunseop; Nam, Kweon-Ho; Paeng, Dong-Guk; Lee, Sang-Joon

2014-08-01

Ultrasound speckle image of blood is mainly attributed by red blood cells (RBCs) which tend to form RBC aggregates. RBC aggregates are separated into individual cells when the shear force is over a certain value. The dissociation of RBC aggregates has an influence on the performance of ultrasound speckle image velocimetry (SIV) technique in which a cross-correlation algorithm is applied to the speckle images to get the velocity field information. The present study aims to investigate the effect of the dissociation of RBC aggregates on the estimation quality of SIV technique. Ultrasound B-mode images were captured from the porcine blood circulating in a mock-up flow loop with varying flow rate. To verify the measurement performance of SIV technique, the centerline velocity measured by the SIV technique was compared with that measured by Doppler spectrograms. The dissociation of RBC aggregates was estimated by using decorrelation of speckle patterns in which the subsequent window was shifted as much as the speckle displacement to compensate decorrelation caused by in-plane loss of speckle patterns. The decorrelation of speckles is considerably increased according to shear rate. Its variations are different along the radial direction. Because the dissociation of RBC aggregates changes ultrasound speckles, the estimation quality of SIV technique is significantly correlated with the decorrelation of speckles. This degradation of measurement quality may be improved by increasing the data acquisition rate. This study would be useful for simultaneous measurement of hemodynamic and hemorheological information of blood flows using only speckle images. Copyright © 2014 Elsevier B.V. All rights reserved.

Chronic psoriatic skin inflammation leads to increased monocyte adhesion and aggregation

PubMed Central

Golden, Jackelyn B.; Groft, Sarah G.; Squeri, Michael V.; Debanne, Sara M.; Ward, Nicole L.; McCormick, Thomas S.; Cooper, Kevin D.

2015-01-01

Psoriasis patients exhibit an increased risk of death by cardiovascular disease (CVD) and have elevated levels of circulating intermediate (CD14++CD16+) monocytes. This elevation could represent evidence of monocyte dysfunction in psoriasis patients at risk of CVD, as increases in circulating CD14++CD16+ monocytes are predictive of myocardial infarction and death. An elevation in the CD14++CD16+ cell population has been previously reported in patients with psoriatic disease, which has been confirmed in the cohort of our human psoriasis patients. CD16 expression was induced in CD14++CD16neg classical monocytes following plastic adhesion, which also elicited enhanced β2 but not β1 integrin surface expression, suggesting increased adhesive capacity. Indeed, we found that psoriasis patients have increased monocyte aggregation among circulating PBMCs which is recapitulated in the KC-Tie2 murine model of psoriasis. Visualization of human monocyte aggregates using imaging cytometry revealed that classical CD14++CD16neg monocytes are the predominant cell type participating in these aggregate pairs. Many of these pairs also included CD16+ monocytes, which could account for apparent elevations of intermediate monocytes. Additionally, intermediate monocytes and monocyte aggregates were the predominant cell type to adhere to TNF-α and IL-17A-stimulated dermal endothelium. Ingenuity Pathway Analysis (IPA) demonstrated that monocyte aggregates have a distinct transcriptional profile from singlet monocytes and monocytes following plastic adhesion, suggesting that circulating monocyte responses to aggregation are not fully accounted for by homotypic adhesion, and that further factors influence their functionality. PMID:26223654

Identification of New Factors Modulating Adhesion Abilities of the Pioneer Commensal Bacterium Streptococcus salivarius

PubMed Central

Couvigny, Benoit; Kulakauskas, Saulius; Pons, Nicolas; Quinquis, Benoit; Abraham, Anne-Laure; Meylheuc, Thierry; Delorme, Christine; Renault, Pierre; Briandet, Romain; Lapaque, Nicolas; Guédon, Eric

2018-01-01

Biofilm formation is crucial for bacterial community development and host colonization by Streptococcus salivarius, a pioneer colonizer and commensal bacterium of the human gastrointestinal tract. This ability to form biofilms depends on bacterial adhesion to host surfaces, and on the intercellular aggregation contributing to biofilm cohesiveness. Many S. salivarius isolates auto-aggregate, an adhesion process mediated by cell surface proteins. To gain an insight into the genetic factors of S. salivarius that dictate host adhesion and biofilm formation, we developed a screening method, based on the differential sedimentation of bacteria in semi-liquid conditions according to their auto-aggregation capacity, which allowed us to identify twelve mutations affecting this auto-aggregation phenotype. Mutations targeted genes encoding (i) extracellular components, including the CshA surface-exposed protein, the extracellular BglB glucan-binding protein, the GtfE, GtfG and GtfH glycosyltransferases and enzymes responsible for synthesis of cell wall polysaccharides (CwpB, CwpK), (ii) proteins responsible for the extracellular localization of proteins, such as structural components of the accessory SecA2Y2 system (Asp1, Asp2, SecA2) and the SrtA sortase, and (iii) the LiaR transcriptional response regulator. These mutations also influenced biofilm architecture, revealing that similar cell-to-cell interactions govern assembly of auto-aggregates and biofilm formation. We found that BglB, CshA, GtfH and LiaR were specifically associated with bacterial auto-aggregation, whereas Asp1, Asp2, CwpB, CwpK, GtfE, GtfG, SecA2 and SrtA also contributed to adhesion to host cells and host-derived components, or to interactions with the human pathogen Fusobacterium nucleatum. Our study demonstrates that our screening method could also be used to identify genes implicated in the bacterial interactions of pathogens or probiotics, for which aggregation is either a virulence trait or an advantageous feature, respectively. PMID:29515553

Aggregation and lack of secretion of most newly synthesized proinsulin in non-beta-cell lines.

PubMed

Zhu, Yong Lian; Abdo, Alexander; Gesmonde, Joan F; Zawalich, Kathleen C; Zawalich, Walter; Dannies, Priscilla S

2004-08-01

Myoblasts transfected with HB10D insulin secrete more hormone than those transfected with wild-type insulin, as published previously, indicating that production of wild-type insulin is not efficient in these cells. The ability of non-beta-cells to produce insulin was examined in several cell lines. In clones of neuroendocrine GH(4)C(1) cells stably transfected with proinsulin, two thirds of (35)S-proinsulin was degraded within 3 h of synthesis, whereas (35)S-prolactin was stable. In transiently transfected neuroendocrine AtT20 cells, half of (35)S-proinsulin was degraded within 3 h after synthesis, whereas (35)S-GH was stable. In transiently transfected fibroblast COS cells, (35)S-proinsulin was stable for longer, but less than 10% was secreted 8 h after synthesis. Proinsulin formed a concentrated patch detected by immunofluorescence in transfected cells that did not colocalize with calreticulin or BiP, markers for the endoplasmic reticulum, but did colocalize with membrin, a marker for the cis-medial Golgi complex. Proinsulin formed a Lubrol-insoluble aggregate within 30 min after synthesis in non-beta-cells but not in INS-1E cells, a beta-cell line that normally produces insulin. More than 45% of (35)S-HB10D proinsulin was secreted from COS cells 3 h after synthesis, and this mutant formed less Lubrol-insoluble aggregate in the cells than did wild-type hormone. These results indicate that proinsulin production from these non-beta-cells is not efficient and that proinsulin aggregates in their secretory pathways. Factors in the environment of the secretory pathway of beta-cells may prevent aggregation of proinsulin to allow efficient production.

AgHalo: A Facile Fluorogenic Sensor to Detect Drug-Induced Proteome Stress.

PubMed

Liu, Yu; Fares, Matthew; Dunham, Noah P; Gao, Zi; Miao, Kun; Jiang, Xueyuan; Bollinger, Samuel S; Boal, Amie K; Zhang, Xin

2017-07-17

Drug-induced proteome stress that involves protein aggregation may cause adverse effects and undermine the safety profile of a drug. Safety of drugs is regularly evaluated using cytotoxicity assays that measure cell death. However, these assays provide limited insights into the presence of proteome stress in live cells. A fluorogenic protein sensor is reported to detect drug-induced proteome stress prior to cell death. An aggregation prone Halo-tag mutant (AgHalo) was evolved to sense proteome stress through its aggregation. Detection of such conformational changes was enabled by a fluorogenic ligand that fluoresces upon AgHalo forming soluble aggregates. Using 5 common anticancer drugs, we exemplified detection of differential proteome stress before any cell death was observed. Thus, this sensor can be used to evaluate drug safety in a regime that the current cytotoxicity assays cannot cover and be generally applied to detect proteome stress induced by other toxins. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Kinetic Monte Carlo and cellular particle dynamics simulations of multicellular systems

NASA Astrophysics Data System (ADS)

Flenner, Elijah; Janosi, Lorant; Barz, Bogdan; Neagu, Adrian; Forgacs, Gabor; Kosztin, Ioan

2012-03-01

Computer modeling of multicellular systems has been a valuable tool for interpreting and guiding in vitro experiments relevant to embryonic morphogenesis, tumor growth, angiogenesis and, lately, structure formation following the printing of cell aggregates as bioink particles. Here we formulate two computer simulation methods: (1) a kinetic Monte Carlo (KMC) and (2) a cellular particle dynamics (CPD) method, which are capable of describing and predicting the shape evolution in time of three-dimensional multicellular systems during their biomechanical relaxation. Our work is motivated by the need of developing quantitative methods for optimizing postprinting structure formation in bioprinting-assisted tissue engineering. The KMC and CPD model parameters are determined and calibrated by using an original computational-theoretical-experimental framework applied to the fusion of two spherical cell aggregates. The two methods are used to predict the (1) formation of a toroidal structure through fusion of spherical aggregates and (2) cell sorting within an aggregate formed by two types of cells with different adhesivities.

Cholesterol impairment contributes to neuroserpin aggregation

NASA Astrophysics Data System (ADS)

Giampietro, Costanza; Lionetti, Maria Chiara; Costantini, Giulio; Mutti, Federico; Zapperi, Stefano; La Porta, Caterina A. M.

2017-03-01

Intraneural accumulation of misfolded proteins is a common feature of several neurodegenerative pathologies including Alzheimer’s and Parkinson’s diseases, and Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB). FENIB is a rare disease due to a point mutation in neuroserpin which accelerates protein aggregation in the endoplasmic reticulum (ER). Here we show that cholesterol depletion induced either by prolonged exposure to statins or by inhibiting the sterol reg-ulatory binding-element protein (SREBP) pathway also enhances aggregation of neuroserpin proteins. These findings can be explained considering a computational model of protein aggregation under non-equilibrium conditions, where a decrease in the rate of protein clearance improves aggregation. Decreasing cholesterol in cell membranes affects their biophysical properties, including their ability to form the vesicles needed for protein clearance, as we illustrate by a simple mathematical model. Taken together, these results suggest that cholesterol reduction induces neuroserpin aggregation, even in absence of specific neuroserpin mutations. The new mechanism we uncover could be relevant also for other neurodegenerative diseases associated with protein aggregation.

Signature of an aggregation-prone conformation of tau

NASA Astrophysics Data System (ADS)

Eschmann, Neil A.; Georgieva, Elka R.; Ganguly, Pritam; Borbat, Peter P.; Rappaport, Maxime D.; Akdogan, Yasar; Freed, Jack H.; Shea, Joan-Emma; Han, Songi

2017-03-01

The self-assembly of the microtubule associated tau protein into fibrillar cell inclusions is linked to a number of devastating neurodegenerative disorders collectively known as tauopathies. The mechanism by which tau self-assembles into pathological entities is a matter of much debate, largely due to the lack of direct experimental insights into the earliest stages of aggregation. We present pulsed double electron-electron resonance measurements of two key fibril-forming regions of tau, PHF6 and PHF6*, in transient as aggregation happens. By monitoring the end-to-end distance distribution of these segments as a function of aggregation time, we show that the PHF6(*) regions dramatically extend to distances commensurate with extended β-strand structures within the earliest stages of aggregation, well before fibril formation. Combined with simulations, our experiments show that the extended β-strand conformational state of PHF6(*) is readily populated under aggregating conditions, constituting a defining signature of aggregation-prone tau, and as such, a possible target for therapeutic interventions.

Aggregates assembled from overexpression of wild-type alpha-synuclein are not toxic to human neuronal cells.

PubMed

Ko, Li-Wen; Ko, Hwai-Hwa C; Lin, Wen-Lang; Kulathingal, Jayanranyan G; Yen, Shu-Hui C

2008-11-01

Filamentous alpha-synuclein (alpha-syn) aggregates form Lewy bodies (LBs), the neuropathologic hallmarks of Parkinson disease and related alpha-synucleinopathies. To model Lewy body-associated neurodegeneration, we generated transfectant 3D5 of human neuronal-type in which expression of human wild-type alpha-syn is regulated by the tetracycline off (TetOff)-inducible mechanism. Retinoic acid-elicited differentiation promoted assembly of alpha-syn aggregates after TetOff induction in 3D5 cells. The aggregates accumulated 14 days after TetOff induction were primarily soluble and showed augmented thioflavin affinity with concomitant phosphorylation and nitration of alpha-syn. Extension of the induction led to the formation of sarkosyl-insoluble aggregates that appeared concurrently with thioflavin-positive inclusions. Immunoelectron microscopy revealed that the inclusions consist of dense bundles of 8- to 12-nm alpha-syn fibrils that congregate in the perikarya and resemble Lewy bodies. Most importantly, accumulation of soluble and insoluble aggregates after TetOff induction for 14 and 28 days was reversible and did not compromise the viability of the cells or their subsequent survival. Thus, this chemically defined culture paradigm provides a useful means to elucidate how oxidative injuries and other insults that are associated with aging promote alpha-syn to self-assemble or interact with other molecules leading to neuronal degeneration in alpha-synucleinopathies.

Role of maternal Xenopus syntabulin in germ plasm aggregation and primordial germ cell specification.

PubMed

Oh, Denise; Houston, Douglas W

2017-12-15

The localization and organization of mitochondria- and ribonucleoprotein granule-rich germ plasm is essential for many aspects of germ cell development. In Xenopus, germ plasm is maternally inherited and is required for the specification of primordial germ cells (PGCs). Germ plasm is aggregated into larger patches during egg activation and cleavage and is ultimately translocated perinuclearly during gastrulation. Although microtubule dynamics and a kinesin (Kif4a) have been implicated in Xenopus germ plasm localization, little is known about how germ plasm distribution is regulated. Here, we identify a role for maternal Xenopus Syntabulin in the aggregation of germ plasm following fertilization. We show that depletion of sybu mRNA using antisense oligonucleotides injected into oocytes results in defects in the aggregation and perinuclear transport of germ plasm and subsequently in reduced PGC numbers. Using live imaging analysis, we also characterize a novel role for Sybu in the collection of germ plasm in vegetal cleavage furrows by surface contraction waves. Additionally, we show that a localized kinesin-like protein, Kif3b, is also required for germ plasm aggregation and that Sybu functionally interacts with Kif3b and Kif4a in germ plasm aggregation. Overall, these data suggest multiple coordinate roles for kinesins and adaptor proteins in controlling the localization and distribution of a cytoplasmic determinant in early development. Copyright © 2017 Elsevier Inc. All rights reserved.

Functional exploration of extracellular polymeric substances (EPS) in the bioleaching of obsolete electric vehicle LiNixCoyMn1-x-yO2 Li-ion batteries.

PubMed

Wang, Jia; Tian, Bingyang; Bao, Yihui; Qian, Can; Yang, Yiran; Niu, Tianqi; Xin, Baoping

2018-07-15

As a fairly new concept, the recovery of valuable metals from urban mining by using bioleaching has become a hotspot. However, the function of extracellular polymeric substances (EPS) in the bioleaching of urban mining gains little attention. The current study used spent EV LIBs to represent urban mining products and systematically explored the function and role of EPS in the attachment of cells to the cathodes, formation of aggregates (cell-EPS-cathode), variation in the electrical and surface properties of the aggregates, concentration of both Fe 2+ and Fe 3+ surrounding the aggregates, electron transfer inside the aggregates and metals released from the aggregates. The results indicated that a strong adhesion of cells to the cathodes occurs mediated by EPS via both hydrophobic force as a main role and electrostatic force as a minor role. Second, the EPS not only adsorb Fe 3+ but also more strongly adsorb Fe 2+ to concentrate the Fe 2+ /Fe 3+ cycle inside the aggregates, witnessing stronger reductive attack on the high valence state of metals as a contact reductive mechanism. Third, the retention or addition of EPS elevated the electronic potential and reduced the electronic resistance to lift the corrosion electric current, thereby boosting the electron transfer and metal dissolution. Copyright © 2018 Elsevier B.V. All rights reserved.

Cellular Inclusion Bodies of Mutant Huntingtin Exon 1 Obscure Small Fibrillar Aggregate Species

PubMed Central

Sahl, Steffen J.; Weiss, Lucien E.; Duim, Whitney C.; Frydman, Judith; Moerner, W. E.

2012-01-01

The identities of toxic aggregate species in Huntington's disease pathogenesis remain ambiguous. While polyQ-expanded huntingtin (Htt) is known to accumulate in compact inclusion bodies inside neurons, this is widely thought to be a protective coping response that sequesters misfolded conformations or aggregated states of the mutated protein. To define the spatial distributions of fluorescently-labeled Htt-exon1 species in the cell model PC12m, we employed highly sensitive single-molecule super-resolution fluorescence imaging. In addition to inclusion bodies and the diffuse pool of monomers and oligomers, fibrillar aggregates ~100 nm in diameter and up to ~1–2 µm in length were observed for pathogenic polyQ tracts (46 and 97 repeats) after targeted photo-bleaching of the inclusion bodies. These short structures bear a striking resemblance to fibers described in vitro. Definition of the diverse Htt structures in cells will provide an avenue to link the impact of therapeutic agents to aggregate populations and morphologies. PMID:23193437

Shaken, but not stirred: how vortical flow drives small-scale aggregations of gyrotactic phytoplankton

NASA Astrophysics Data System (ADS)

Barry, Michael; Durham, William; Climent, Eric; Stocker, Roman

2011-11-01

Coastal ocean observations reveal that motile phytoplankton form aggregations at the Kolmogorov scale (mm-cm), whereas non-motile cells do not. We propose a new mechanism for the formation of this small-scale patchiness based on the interplay of turbulence and gyrotactic motility. Counterintuitively, turbulence does not stir a plankton suspension to homogeneity but drives aggregations instead. Through controlled laboratory experiments we show that the alga Heterosigma akashiwo rapidly forms aggregations in a cavity-driven vortical flow that approximates Kolmogorov eddies. Gyrotactic motility is found to be the key ingredient for aggregation, as non-motile cells remain randomly distributed. Observations are in remarkable agreement with a 3D model, and the validity of this mechanism for generating patchiness has been extended to realistic turbulent flows using Direct Numerical Simulations. Because small-scale patchiness influences rates of predation, sexual reproduction, infection, and nutrient competition, this result indicates that gyrotactic motility can profoundly affect phytoplankton ecology.

Brain-derived exosomes from dementia with Lewy bodies propagate α-synuclein pathology.

PubMed

Ngolab, Jennifer; Trinh, Ivy; Rockenstein, Edward; Mante, Michael; Florio, Jazmin; Trejo, Margarita; Masliah, Deborah; Adame, Anthony; Masliah, Eliezer; Rissman, Robert A

2017-06-09

Proteins implicated in neurodegenerative conditions such as Alzheimer's disease (AD) and Dementia with Lewy Bodies (DLB) have been identified in bodily fluids encased in extracellular vesicles called exosomes. Whether exosomes found in DLB patients can transmit pathology is not clear. In this study, exosomes were successfully harvested through ultracentrifugation from brain tissue from DLB and AD patients as well as non-diseased brain tissue. Exosomes extracted from brains diagnosed with either AD or DLB contained aggregate-prone proteins. Furthermore, injection of brain-derived exosomes from DLB patients into the brains of wild type mice induced α-synuclein (α-syn) aggregation. As assessed through immunofluorescent double labeling, α-syn aggregation was observed in MAP2 + , Rab5 + neurons. Using a neuronal cell line, we also identified intracellular α-syn aggregation mediated by exosomes is dependent on recipient cell endocytosis. Together, these data suggest that exosomes from DLB patients are sufficient for seeding and propagating α-syn aggregation in vivo.

Novel polyglutamine model uncouples proteotoxicity from aging.

PubMed

Christie, Nakeirah T M; Lee, Amy L; Fay, Hannah G; Gray, Amelia A; Kikis, Elise A

2014-01-01

Polyglutamine expansions in certain proteins are the genetic determinants for nine distinct progressive neurodegenerative disorders and resultant age-related dementia. In these cases, neurodegeneration is due to the aggregation propensity and resultant toxic properties of the polyglutamine-containing proteins. We are interested in elucidating the underlying mechanisms of toxicity of the protein ataxin-3, in which a polyglutamine expansion is the genetic determinant for Machado-Joseph Disease (MJD), also referred to as spinocerebellar ataxia 3 (SCA3). To this end, we have developed a novel model for ataxin-3 protein aggregation, by expressing a disease-related polyglutamine-containing fragment of ataxin-3 in the genetically tractable body wall muscle cells of the model system C. elegans. Here, we demonstrate that this ataxin-3 fragment aggregates in a polyQ length-dependent manner in C. elegans muscle cells and that this aggregation is associated with cellular dysfunction. However, surprisingly, this aggregation and resultant toxicity was not influenced by aging. This is in contrast to polyglutamine peptides alone whose aggregation/toxicity is highly dependent on age. Thus, the data presented here not only describe a new polyglutamine model, but also suggest that protein context likely influences the cellular interactions of the polyglutamine-containing protein and thereby modulates its toxic properties.

Solubilization of protein aggregates by the acid stress chaperones HdeA and HdeB.

PubMed

Malki, Abderrahim; Le, Hai-Tuong; Milles, Sigrid; Kern, Renée; Caldas, Teresa; Abdallah, Jad; Richarme, Gilbert

2008-05-16

The acid stress chaperones HdeA and HdeB of Escherichia coli prevent the aggregation of periplasmic proteins at acidic pH. We show in this report that they also form mixed aggregates with proteins that have failed to be solubilized at acidic pH and allow their subsequent solubilization at neutral pH. HdeA, HdeB, and HdeA and HdeB together display an increasing efficiency for the solubilization of protein aggregates at pH 3. They are less efficient for the solubilization of aggregates at pH 2, whereas HdeB is the most efficient. Increasing amounts of periplasmic proteins draw increasing amounts of chaperone into pellets, suggesting that chaperones co-aggregate with their substrate proteins. We observed a decrease in the size of protein aggregates in the presence of HdeA and HdeB, from very high molecular mass aggregates to 100-5000-kDa species. Moreover, a marked decrease in the exposed hydrophobicity of aggregated proteins in the presence of HdeA and HdeB was revealed by 1,1'-bis(4-anilino)naphtalene-5,5'-disulfonic acid binding experiments. In vivo, during the recovery at neutral pH of acid stressed bacterial cells, HdeA and HdeB allow the solubilization and renaturation of protein aggregates, including those formed by the maltose receptor MalE, the oligopeptide receptor OppA, and the histidine receptor HisJ. Thus, HdeA and HdeB not only help to maintain proteins in a soluble state during acid treatment, as previously reported, but also assist, both in vitro and in vivo, in the solubilization at neutral pH of mixed protein-chaperone aggregates formed at acidic pH, by decreasing the size of protein aggregates and the exposed hydrophobicity of aggregated proteins.

Physical Mechanisms Driving Cell Sorting in Hydra.

PubMed

Cochet-Escartin, Olivier; Locke, Tiffany T; Shi, Winnie H; Steele, Robert E; Collins, Eva-Maria S

2017-12-19

Cell sorting, whereby a heterogeneous cell mixture organizes into distinct tissues, is a fundamental patterning process in development. Hydra is a powerful model system for carrying out studies of cell sorting in three dimensions, because of its unique ability to regenerate after complete dissociation into individual cells. The physicists Alfred Gierer and Hans Meinhardt recognized Hydra's self-organizing properties more than 40 years ago. However, what drives cell sorting during regeneration of Hydra from cell aggregates is still debated. Differential motility and differential adhesion have been proposed as driving mechanisms, but the available experimental data are insufficient to distinguish between these two. Here, we answer this longstanding question by using transgenic Hydra expressing fluorescent proteins and a multiscale experimental and numerical approach. By quantifying the kinematics of single cell and whole aggregate behaviors, we show that no differences in cell motility exist among cell types and that sorting dynamics follow a power law with an exponent of ∼0.5. Additionally, we measure the physical properties of separated tissues and quantify their viscosities and surface tensions. Based on our experimental results and numerical simulations, we conclude that tissue interfacial tensions are sufficient to explain cell sorting in aggregates of Hydra cells. Furthermore, we demonstrate that the aggregate's geometry during sorting is key to understanding the sorting dynamics and explains the exponent of the power law behavior. Our results answer the long standing question of the physical mechanisms driving cell sorting in Hydra cell aggregates. In addition, they demonstrate how powerful this organism is for biophysical studies of self-organization and pattern formation. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Equine cloning: in vitro and in vivo development of aggregated embryos.

PubMed

Gambini, Andrés; Jarazo, Javier; Olivera, Ramiro; Salamone, Daniel F

2012-07-01

The production of cloned equine embryos remains highly inefficient. Embryo aggregation has not yet been tested in the equine, and it might represent an interesting strategy to improve embryo development. This study evaluated the effect of cloned embryo aggregation on in vitro and in vivo equine embryo development. Zona-free reconstructed embryos were individually cultured in microwells (nonaggregated group) or as 2- or 3-embryo aggregates (aggregated groups). For in vitro development, they were cultured until blastocyst stage and then either fixed for Oct-4 immunocytochemical staining or maintained in in vitro culture where blastocyst expansion was measured daily until Day 17 or the day on which they collapsed. For in vivo assays, Day 7-8 blastocysts were transferred to synchronized mares and resultant vesicles, and cloned embryos were measured by ultrasonography. Embryo aggregation improved blastocyst rates on a per well basis, and aggregation did not imply additional oocytes to obtain blastocysts. Embryo aggregation improved embryo quality, nevertheless it did not affect Day 8 and Day 16 blastocyst Oct-4 expression patterns. Equine cloned blastocysts expanded and increased their cell numbers when they were maintained in in vitro culture, describing a particular pattern of embryo growth that was unexpectedly independent of embryo aggregation, as all embryos reached similar size after Day 7. Early pregnancy rates were higher using blastocysts derived from aggregated embryos, and advanced pregnancies as live healthy foals also resulted from aggregated embryos. These results indicate that the strategy of aggregating embryos can improve their development, supporting the establishment of equine cloned pregnancies.

Putative porcine embryonic stem cell lines derived from aggregated four-celled cloned embryos produced by oocyte bisection cloning.

PubMed

Siriboon, Chawalit; Lin, Yu-Hsuan; Kere, Michel; Chen, Chun-Da; Chen, Lih-Ren; Chen, Chien-Hong; Tu, Ching-Fu; Lo, Neng-Wen; Ju, Jyh-Cherng

2015-01-01

We attempted to isolate ES cell lines using inner cell masses from high-quality cloned porcine blastocysts. After being seeded onto feeders, embryos had better (P < 0.05) attachment, outgrowth formation and primary colonization in both 2× and 3× aggregated cloned embryos (62.8, 42.6 and 12.8% vs. 76.2, 55.2 and 26.2%, respectively) compared to the non-aggregated group (41.6, 23.4 and 3.9%). Effects of feeder types (STO vs. MEF) and serum sources (FBS vs. KSR) on extraction of cloned embryo-derived porcine ES cells were examined. More (17.1%) ntES cell lines over Passage 3 were generated in the MEF/KSR group. However, ntES cells cultured in KSR-supplemented medium had a low proliferation rate with defective morphology, and eventually underwent differentiation or apoptosis subsequently. Approximately 26.1, 22.7 and 35.7% of primary colonies were formed after plating embryos in DMEM, DMEM/F12 and α-MEM media, respectively. Survival rates of ntES cells cultured in α-MEM, DMEM and DMEM/F12 were 16.7, 4.3 and 6.8%, respectively (P > 0.05). We further examined the beneficial effect of TSA treatment of 3× aggregated cloned embryos on establishment of ntES cell lines. Primary colony numbers and survival rates of ntES cells beyond passage 3 were higher (P < 0.05) in those derived from TSA-treated 3× blastocysts (36.7 and 26.7%) than from the non-treated aggregated group (23.1 and 11.5%). These cells, remaining undifferentiated over 25 passages, had alkaline phosphatase activity and expressed ES specific markers Oct4, Nanog, Sox2, and Rex01. Moreover, these ntES cells successfully differentiated into embryoid bodies (EBs) that expressed specific genes of all three germ layers after being cultured in LIF-free medium. In conclusion, we have successfully derived putative porcine ntES cells with high efficiency from quality cloned embryos produced by embryo aggregation, and optimized the ES cell culture system suitable for establishing and maintaining ntES cell lines in undifferentiated state.

Putative Porcine Embryonic Stem Cell Lines Derived from Aggregated Four-Celled Cloned Embryos Produced by Oocyte Bisection Cloning

PubMed Central

Siriboon, Chawalit; Lin, Yu-Hsuan; Kere, Michel; Chen, Chun-Da; Chen, Lih-Ren; Chen, Chien-Hong; Tu, Ching-Fu; Lo, Neng-Wen; Ju, Jyh-Cherng

2015-01-01

We attempted to isolate ES cell lines using inner cell masses from high-quality cloned porcine blastocysts. After being seeded onto feeders, embryos had better (P < 0.05) attachment, outgrowth formation and primary colonization in both 2× and 3× aggregated cloned embryos (62.8, 42.6 and12.8% vs. 76.2, 55.2 and 26.2%, respectively) compared to the non-aggregated group (41.6, 23.4 and 3.9%). Effects of feeder types (STO vs. MEF) and serum sources (FBS vs. KSR) on extraction of cloned embryo-derived porcine ES cells were examined. More (17.1%) ntES cell lines over Passage 3 were generated in the MEF/KSR group. However, ntES cells cultured in KSR-supplemented medium had a low proliferation rate with defective morphology, and eventually underwent differentiation or apoptosis subsequently. Approximately 26.1, 22.7 and 35.7% of primary colonies were formed after plating embryos in DMEM, DMEM/F12 and α-MEM media, respectively. Survival rates of ntES cells cultured in α-MEM, DMEM and DMEM/F12 were 16.7, 4.3 and 6.8%, respectively (P > 0.05). We further examined the beneficial effect of TSA treatment of 3× aggregated cloned embryos on establishment of ntES cell lines. Primary colony numbers and survival rates of ntES cells beyond passage 3 were higher (P < 0.05) in those derived from TSA-treated 3× blastocysts (36.7 and 26.7%) than from the non-treated aggregated group (23.1 and 11.5%). These cells, remaining undifferentiated over 25 passages, had alkaline phosphatase activity and expressed ES specific markers Oct4, Nanog, Sox2, and Rex01. Moreover, these ntES cells successfully differentiated into embryoid bodies (EBs) that expressed specific genes of all three germ layers after being cultured in LIF-free medium. In conclusion, we have successfully derived putative porcine ntES cells with high efficiency from quality cloned embryos produced by embryo aggregation, and optimized the ES cell culture system suitable for establishing and maintaining ntES cell lines in undifferentiated state. PMID:25680105

Ubiquilin overexpression reduces GFP-polyalanine-induced protein aggregates and toxicity

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

Wang Hongmin; Monteiro, Mervyn J.

2007-08-01

Several human disorders are associated with an increase in a continuous stretch of alanine amino acids in proteins. These so-called polyalanine expansion diseases share many similarities with polyglutamine-related disorders, including a length-dependent reiteration of amino acid induction of protein aggregation and cytotoxicity. We previously reported that overexpression of ubiquilin reduces protein aggregates and toxicity of expanded polyglutamine proteins. Here, we demonstrate a similar role for ubiquilin toward expanded polyalanine proteins. Overexpression of ubiquilin-1 in HeLa cells reduced protein aggregates and the cytotoxicity associated with expression of a transfected nuclear-targeted GFP-fusion protein containing 37-alanine repeats (GFP-A37), in a dose dependent manner.more » Ubiquilin coimmunoprecipitated more with GFP proteins containing a 37-polyalanine tract compared to either 7 (GFP-A7), or no alanine tract (GFP). Moreover, overexpression of ubiquilin suppressed the increased vulnerability of HeLa cell lines stably expressing the GFP-A37 fusion protein to oxidative stress-induced cell death compared to cell lines expressing GFP or GFP-A7 proteins. By contrast, siRNA knockdown of ubiquilin expression in the GFP-A37 cell line was associated with decreased cellular proliferation, and increases in GFP protein aggregates, nuclear fragmentation, and cell death. Our results suggest that boosting ubiquilin levels in cells might provide a universal and attractive strategy to prevent toxicity of proteins containing reiterative expansions of amino acids involved in many human diseases.« less

Differential toxicity, conformation and morphology of typical initial aggregation states of Aβ1-42 and Aβpy3-42 beta-amyloids.

PubMed

Galante, Denise; Corsaro, Alessandro; Florio, Tullio; Vella, Serena; Pagano, Aldo; Sbrana, Francesca; Vassalli, Massimo; Perico, Angelo; D'Arrigo, Cristina

2012-11-01

Among the different species of water-soluble β-peptides (Aβ1-42, Aβ1-40 and N-terminal truncated Aβ-peptides), Aβpy3-42 is thought to play a relevant role in Alzheimer's pathogenesis due to its abundance, resistance to proteolysis, fast aggregation kinetics, dynamic structure and high neurotoxicity. To evaluate the specific structural characteristics and neurotoxicity of Aβpy3-42, we separated different aggregation states of Aβ1-42 and Aβpy3-42 using fast protein liquid chromatography, isolating in both cases three peaks that corresponded to sa (small), ma (medium) and la (large) aggregates. Conformational analysis, by circular dichroism showed a prevailing random coil conformation for sa and ma, and typical β-sheet conformation for la. AFM and TEM show differential structural features between the three aggregates of a given β-peptide and among the aggregate of the two β-peptides. The potential toxic effects of the different aggregates were evaluated using human neuroblastoma SH-SY5Y cells in the MTT reduction, in the xCELLigence System, and in the Annexin V binding experiments. In the case of Aβ1-42 the most toxic aggregate is la, while in the case of Aβpy3-42 both sa and la are equally toxic. Aβ aggregates were found to be internalized in the cells, as estimated by confocal immunofluorescence microscopy, with a higher effect observed for Aβpy3-42, showing a good correlation with the toxic effects. Together these experiments allowed the discrimination of the intermediate states more responsible of oligomer toxicity, providing new insights on the correlation between the aggregation process and the toxicity and confirming the peculiar role in the pathogenesis of Alzheimer disease of Aβpy3-42 peptide. Copyright © 2012 Elsevier Ltd. All rights reserved.

Sequestration of the Aβ Peptide Prevents Toxicity and Promotes Degradation In Vivo

PubMed Central

de Barros, Teresa Pereira; van Dijk Härd, Iris; Brorsson, Ann-Christin; Macao, Bertil; Persson, Cecilia; Crowther, Damian C.; Lomas, David A.; Ståhl, Stefan; Dobson, Christopher M.; Härd, Torleif

2010-01-01

Protein aggregation, arising from the failure of the cell to regulate the synthesis or degradation of aggregation-prone proteins, underlies many neurodegenerative disorders. However, the balance between the synthesis, clearance, and assembly of misfolded proteins into neurotoxic aggregates remains poorly understood. Here we study the effects of modulating this balance for the amyloid-beta (Aβ) peptide by using a small engineered binding protein (ZAβ3) that binds with nanomolar affinity to Aβ, completely sequestering the aggregation-prone regions of the peptide and preventing its aggregation. Co-expression of ZAβ3 in the brains of Drosophila melanogaster expressing either Aβ42 or the aggressive familial associated E22G variant of Aβ42 abolishes their neurotoxic effects. Biochemical analysis indicates that monomer Aβ binding results in degradation of the peptide in vivo. Complementary biophysical studies emphasize the dynamic nature of Aβ aggregation and reveal that ZAβ3 not only inhibits the initial association of Aβ monomers into oligomers or fibrils, but also dissociates pre-formed oligomeric aggregates and, although very slowly, amyloid fibrils. Toxic effects of peptide aggregation in vivo can therefore be eliminated by sequestration of hydrophobic regions in monomeric peptides, even when these are extremely aggregation prone. Our studies also underline how a combination of in vivo and in vitro experiments provide mechanistic insight with regard to the relationship between protein aggregation and clearance and show that engineered binding proteins may provide powerful tools with which to address the physiological and pathological consequences of protein aggregation. PMID:20305716

Environmental and genetic factors support the dissociation between α-synuclein aggregation and toxicity.

PubMed

Villar-Piqué, Anna; Lopes da Fonseca, Tomás; Sant'Anna, Ricardo; Szegö, Éva Mónika; Fonseca-Ornelas, Luis; Pinho, Raquel; Carija, Anita; Gerhardt, Ellen; Masaracchia, Caterina; Abad Gonzalez, Enrique; Rossetti, Giulia; Carloni, Paolo; Fernández, Claudio O; Foguel, Debora; Milosevic, Ira; Zweckstetter, Markus; Ventura, Salvador; Outeiro, Tiago Fleming

2016-10-18

Synucleinopathies are a group of progressive disorders characterized by the abnormal aggregation and accumulation of α-synuclein (aSyn), an abundant neuronal protein that can adopt different conformations and biological properties. Recently, aSyn pathology was shown to spread between neurons in a prion-like manner. Proteins like aSyn that exhibit self-propagating capacity appear to be able to adopt different stable conformational states, known as protein strains, which can be modulated both by environmental and by protein-intrinsic factors. Here, we analyzed these factors and found that the unique combination of the neurodegeneration-related metal copper and the pathological H50Q aSyn mutation induces a significant alteration in the aggregation properties of aSyn. We compared the aggregation of WT and H50Q aSyn with and without copper, and assessed the effects of the resultant protein species when applied to primary neuronal cultures. The presence of copper induces the formation of structurally different and less-damaging aSyn aggregates. Interestingly, these aggregates exhibit a stronger capacity to induce aSyn inclusion formation in recipient cells, which demonstrates that the structural features of aSyn species determine their effect in neuronal cells and supports a lack of correlation between toxicity and inclusion formation. In total, our study provides strong support in favor of the hypothesis that protein aggregation is not a primary cause of cytotoxicity.

Environmental and genetic factors support the dissociation between α-synuclein aggregation and toxicity

PubMed Central

Villar-Piqué, Anna; Lopes da Fonseca, Tomás; Sant’Anna, Ricardo; Szegö, Éva Mónika; Fonseca-Ornelas, Luis; Pinho, Raquel; Carija, Anita; Gerhardt, Ellen; Masaracchia, Caterina; Abad Gonzalez, Enrique; Rossetti, Giulia; Carloni, Paolo; Fernández, Claudio O.; Foguel, Debora; Milosevic, Ira; Zweckstetter, Markus; Ventura, Salvador; Outeiro, Tiago Fleming

2016-01-01

Synucleinopathies are a group of progressive disorders characterized by the abnormal aggregation and accumulation of α-synuclein (aSyn), an abundant neuronal protein that can adopt different conformations and biological properties. Recently, aSyn pathology was shown to spread between neurons in a prion-like manner. Proteins like aSyn that exhibit self-propagating capacity appear to be able to adopt different stable conformational states, known as protein strains, which can be modulated both by environmental and by protein-intrinsic factors. Here, we analyzed these factors and found that the unique combination of the neurodegeneration-related metal copper and the pathological H50Q aSyn mutation induces a significant alteration in the aggregation properties of aSyn. We compared the aggregation of WT and H50Q aSyn with and without copper, and assessed the effects of the resultant protein species when applied to primary neuronal cultures. The presence of copper induces the formation of structurally different and less-damaging aSyn aggregates. Interestingly, these aggregates exhibit a stronger capacity to induce aSyn inclusion formation in recipient cells, which demonstrates that the structural features of aSyn species determine their effect in neuronal cells and supports a lack of correlation between toxicity and inclusion formation. In total, our study provides strong support in favor of the hypothesis that protein aggregation is not a primary cause of cytotoxicity. PMID:27708160

Cluster-cluster aggregation with particle replication and chemotaxy: a simple model for the growth of animal cells in culture

NASA Astrophysics Data System (ADS)

Alves, S. G.; Martins, M. L.

2010-09-01

Aggregation of animal cells in culture comprises a series of motility, collision and adhesion processes of basic relevance for tissue engineering, bioseparations, oncology research and in vitro drug testing. In the present paper, a cluster-cluster aggregation model with stochastic particle replication and chemotactically driven motility is investigated as a model for the growth of animal cells in culture. The focus is on the scaling laws governing the aggregation kinetics. Our simulations reveal that in the absence of chemotaxy the mean cluster size and the total number of clusters scale in time as stretched exponentials dependent on the particle replication rate. Also, the dynamical cluster size distribution functions are represented by a scaling relation in which the scaling function involves a stretched exponential of the time. The introduction of chemoattraction among the particles leads to distribution functions decaying as power laws with exponents that decrease in time. The fractal dimensions and size distributions of the simulated clusters are qualitatively discussed in terms of those determined experimentally for several normal and tumoral cell lines growing in culture. It is shown that particle replication and chemotaxy account for the simplest cluster size distributions of cellular aggregates observed in culture.

Influence of mechanical cell salvage on red blood cell aggregation, deformability, and 2,3-diphosphoglycerate in patients undergoing cardiac surgery with cardiopulmonary bypass.

PubMed

Gu, Y John; Vermeijden, Wytze J; de Vries, Adrianus J; Hagenaars, J Ans M; Graaff, Reindert; van Oeveren, Willem

2008-11-01

Mechanical cell salvage is increasingly used during cardiac surgery. Although this procedure is considered safe, it is unknown whether it affects the red blood cell (RBC) function, especially the RBC aggregation, deformability, and the contents of 2,3-diphosphoglycerate (2,3-DPG). This study examines the following: (1) whether the cell salvage procedure influences RBC function; and (2) whether retransfusion of the salvaged blood affects RBC function in patients. Forty patients undergoing cardiac surgery with cardiopulmonary bypass were randomly allocated to a cell saver group (n = 20) or a control group (n = 20). In the cell saver group, the blood aspirated from the wound area and the residual blood from the heart-lung machine were processed with a continuous-flow cell saver before retransfusion. In the control group this blood was retransfused without processing. The RBC aggregation and deformability were measured with a laser-assisted optical rotational cell analyzer and 2,3,-DPG by conventional laboratory test. The cell saver procedure did not influence the RBC aggregation but significantly reduced the RBC deformability (p = 0.007) and the content of RBC 2,3-DPG (p = 0.032). However, in patients receiving the processed blood, their intraoperative and postoperative RBC aggregation, deformability, and 2,3-DPG content did not differ from those of the control patients. Both groups of patients had a postoperative drop of RBC function as a result of hemodilution. The mechanical cell salvage procedure reduces the RBC deformability and the cell 2,3-DPG content. Retransfusion of the processed blood by cell saver does not further compromise the RBC function in patients undergoing cardiac surgery with cardiopulmonary bypass.

Sequential development of apical-basal and planar polarities in aggregating epitheliomuscular cells of Hydra.

PubMed

Seybold, Anna; Salvenmoser, Willi; Hobmayer, Bert

2016-04-01

Apical-basal and planar cell polarities are hallmarks of metazoan epithelia required to separate internal and external environments and to regulate trans- and intracellular transport, cytoskeletal organization, and morphogenesis. Mechanisms of cell polarization have been intensively studied in bilaterian model organisms, particularly in early embryos and cultured cells, while cell polarity in pre-bilaterian tissues is poorly understood. Here, we have studied apical-basal and planar polarization in regenerating (aggregating) clusters of epitheliomuscular cells of Hydra, a simple representative of the ancestral, pre-bilaterian phylum Cnidaria. Immediately after dissociation, single epitheliomuscular cells do not exhibit cellular polarity, but they polarize de novo during aggregation. Reestablishment of the Hydra-specific epithelial bilayer is a result of short-range cell sorting. In the early phase of aggregation, apical-basal polarization starts with an enlargement of the epithelial apical-basal diameter and by the development of belt-like apical septate junctions. Specification of the basal pole of epithelial cells occurs shortly later and is linked to synthesis of mesoglea, development of hemidesmosome-like junctions, and formation of desmosome-like junctions connecting the basal myonemes of neighbouring cells. Planar polarization starts, while apical-basal polarization is already ongoing. It is executed gradually starting with cell-autonomous formation, parallelization, and condensation of myonemes at the basal end of each epithelial cell and continuing with a final planar alignment of epitheliomuscular cells at the tissue level. Our findings reveal that epithelial polarization in Hydra aggregates occurs in defined steps well accessible by histological and ultrastructural techniques and they will provide a basis for future molecular studies. Copyright © 2016 Elsevier Inc. All rights reserved.

Chaperone protein HYPK interacts with the first 17 amino acid region of Huntingtin and modulates mutant HTT-mediated aggregation and cytotoxicity

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

Choudhury, Kamalika Roy; Centre for Neuroscience, Indian Institute of Science, Bangalore 560012; Bhattacharyya, Nitai P., E-mail: nitai_sinp@yahoo.com

2015-01-02

Highlights: • HYPK reduces mutant HTT-mediated aggregate formation and cytotoxicity. • Interaction of HYPK with HTT requires N-terminal 17 amino acid of HTT (HTT-N17). • Deletion of HTT-N17 leads to SDS-soluble, smaller, nuclear aggregates. • These smaller aggregates do not associate with HYPK and are more cytotoxic. • Maybe, interaction of HYPK with amphipathic HTT-N17 block HTT aggregate formation. - Abstract: Huntington’s disease is a polyglutamine expansion disorder, characterized by mutant HTT-mediated aggregate formation and cytotoxicity. Many reports suggests roles of N-terminal 17 amino acid domain of HTT (HTT-N17) towards subcellular localization, aggregate formation and subsequent pathogenicity induced by N-terminalmore » HTT harboring polyQ stretch in pathogenic range. HYPK is a HTT-interacting chaperone which can reduce N-terminal mutant HTT-mediated aggregate formation and cytotoxicity in neuronal cell lines. However, how HYPK interacts with N-terminal fragment of HTT remained unknown. Here we report that specific interaction of HYPK with HTT-N17 is crucial for the chaperone activity of HYPK. Deletion of HTT-N17 leads to formation of tinier, SDS-soluble nuclear aggregates formed by N-terminal mutant HTT. The increased cytotoxicity imparted by these tiny aggregates might be contributed due to loss of interaction with HYPK.« less

Passive participation of fixed platelets in aggregation facilitated by covalently bound fibrinogen.

PubMed

Agam, G; Livne, A

1983-01-01

The role of fibrinogen in interplatelet recognition during aggregation was examined by combining two cell types: fresh platelets (in limiting density) activated by thrombin or A23187, and formaldehyde-fixed platelets, bearing cross-linked fibrinogen. The fixed platelets did not aggregate by themselves, nor with resting platelets, but were capable of interacting with activated platelets and of participating passively in aggregation. The participation, expressed by enhanced aggregation, was assayed by the conventional turbidometric traces and by cosedimentation of fixed 3H-platelets with aggregates of fresh platelets. Platelet suspensions, prepared without special means to avert spontaneous activation, retained plasma fibrinogen to the extent of 50 micrograms/ml of a suspension containing 10(8) platelets, and the derived fixed platelets participated in aggregation, independently of added fibrinogen. The capability of such fixed platelets to participate in aggregation was sensitive to proteolytic digestion and to massive acetylation. When platelet separation was aided by apyrase or aspirin, PGE1 and gel filtration, the residual plasma fibrinogen was limited to 0.4 micrograms/ml of 10(8) platelet suspension. The derived fixed platelets were incapable of participating in aggregation unless fibrinogen was added prior to fixation. The affixed fibrinogen could not be replaced by soluble fibrinogen or affixed albumin. It is concluded that fibrinogen, which binds to platelets upon activation or is linked to them covalently, is a recognition site for platelet-platelet interaction during aggregation.

Familial aggregation analysis of gene expressions

PubMed Central

Rao, Shao-Qi; Xu, Liang-De; Zhang, Guang-Mei; Li, Xia; Li, Lin; Shen, Gong-Qing; Jiang, Yang; Yang, Yue-Ying; Gong, Bin-Sheng; Jiang, Wei; Zhang, Fan; Xiao, Yun; Wang, Qing K

2007-01-01

Traditional studies of familial aggregation are aimed at defining the genetic (and non-genetic) causes of a disease from physiological or clinical traits. However, there has been little attempt to use genome-wide gene expressions, the direct phenotypic measures of genes, as the traits to investigate several extended issues regarding the distributions of familially aggregated genes on chromosomes or in functions. In this study we conducted a genome-wide familial aggregation analysis by using the in vitro cell gene expressions of 3300 human autosome genes (Problem 1 data provided to Genetic Analysis Workshop 15) in order to answer three basic genetics questions. First, we investigated how gene expressions aggregate among different types (degrees) of relative pairs. Second, we conducted a bioinformatics analysis of highly familially aggregated genes to see how they are distributed on chromosomes. Third, we performed a gene ontology enrichment test of familially aggregated genes to find evidence to support their functional consensus. The results indicated that 1) gene expressions did aggregate in families, especially between sibs. Of 3300 human genes analyzed, there were a total of 1105 genes with one or more significant (empirical p < 0.05) familial correlation; 2) there were several genomic hot spots where highly familially aggregated genes (e.g., the chromosome 6 HLA genes cluster) were clustered; 3) as we expected, gene ontology enrichment tests revealed that the 1105 genes were aggregating not only in families but also in functional categories. PMID:18466548

Low-velocity collision behaviour of clusters composed of sub-millimetre sized dust aggregates

NASA Astrophysics Data System (ADS)

Brisset, J.; Heißelmann, D.; Kothe, S.; Weidling, R.; Blum, J.

2017-07-01

Context. The experiment results presented apply to the very first stages of planet formation, when small dust aggregates collide in the protoplanetary disc and grow into bigger clusters. In 2011, before flying on the REXUS 12 suborbital rocket in 2012, the Suborbital Particle and Aggregation Experiment (SPACE) performed drop tower flights. We present the results of this first microgravity campaign. Aims: The experiments presented aim to measure the outcome of collisions between sub-mm sized protoplanetary dust aggregate analogues. We also observed the clusters formed from these aggregates and their collision behaviour. Methods: The experiments were performed at the drop tower in Bremen. The protoplanetary dust analogue materials were micrometre-sized monodisperse and polydisperse SiO2 particles prepared into aggregates with sizes between 120 μm and 250 μm. One of the dust samples contained aggregates that were previously compacted through repeated bouncing. During three flights of 9 s of microgravity each, individual collisions between aggregates and the formation of clusters of up to a few millimetres in size were observed. In addition, the collisions of clusters with the experiment cell walls leading to compaction or fragmentation were recorded. Results: We observed collisions amongst dust aggregates and collisions between dust clusters and the cell aluminium walls at speeds ranging from about 0.1 cm s-1 to 20 cm s-1. The velocities at which sticking occurred ranged from 0.18 to 5.0 cm s-1 for aggregates composed of monodisperse dust, with an average value of 2.1 ± 0.9 cm s-1 for reduced masses ranging from 1.2 × 10-6 to 1.8 × 10-3 g with an average value of 2.2+16-2.1 × 10-4 g. The velocities at which bouncing occurred ranged from 1.9 to 11.9 cm s-1 for the same aggregates with an average of 5.9 ± 3.2 cm s-1 for reduced masses ranging from 2.1 × 10-6 to 2.4 × 10-4 with an average of 7.8 ± 2.4 × 10-5 g. The velocities at which fragmentation occurred ranged from 4.9 to 23.8 cm s-1 for the same aggregates with an average of 10.1 ± 3.2 cm s-1 for reduced masses ranging from 1.2 × 10-5 to 1.2 × 10-3 with an average value of 4.2 ± 2.4 × 10-4 g. From the restructuring and fragmentation of clusters composed of dust aggregates colliding with the aluminium cell walls, we derived a collision recipe for dust aggregates ( 100 μm) following the model of Dominik & Tielens (1997, ApJ, 480, 647) developed for microscopic particles. We measured a critical rolling energy of 1.8 ± 0.9 × 10-13 J and a critical breaking energy of 3.5 ± 1.5 × 10-13 J for 100 μm-sized non-compacted aggregates. A movie associated to Fig. 3 is available at http://www.aanda.org

Effects of Grape Xylem Sap and Cell-Wall Constituents on In Vitro Growth, Biofilm Formation and Cellular Aggregation of Xylella fastidiosa

USDA-ARS?s Scientific Manuscript database

Purified cell-wall constituents or grape xylem sap added to media affected in vitro growth, biofilm formation, cell aggregation and gene expression of Xylella fastidiosa. Media containing xylem sap from Pierce’s disease (PD)-susceptible plants provided better support for bacterial growth and biofil...

Embryos aggregation improves development and imprinting gene expression in mouse parthenogenesis.

PubMed

Bai, Guang-Yu; Song, Si-Hang; Wang, Zhen-Dong; Shan, Zhi-Yan; Sun, Rui-Zhen; Liu, Chun-Jia; Wu, Yan-Shuang; Li, Tong; Lei, Lei

2016-04-01

Mouse parthenogenetic embryonic stem cells (PgESCs) could be applied to study imprinting genes and are used in cell therapy. Our previous study found that stem cells established by aggregation of two parthenogenetic embryos at 8-cell stage (named as a2 PgESCs) had a higher efficiency than that of PgESCs, and the paternal expressed imprinting genes were observably upregulated. Therefore, we propose that increasing the number of parthenogenetic embryos in aggregation may improve the development of parthenogenetic mouse and imprinting gene expression of PgESCs. To verify this hypothesis, we aggregated four embryos together at the 4-cell stage and cultured to the blastocyst stage (named as 4aPgB). qPCR detection showed that the expression of imprinting genes Igf2, Mest, Snrpn, Igf2r, H19, Gtl2 in 4aPgB were more similar to that of fertilized blastocyst (named as fB) compared to 2aPgB (derived from two 4-cell stage parthenogenetic embryos aggregation) or PgB (single parthenogenetic blastocyst). Post-implantation development of 4aPgB extended to 11 days of gestation. The establishment efficiency of GFP-a4 PgESCs which derived from GFP-4aPgB is 62.5%. Moreover, expression of imprinting genes Igf2, Mest, Snrpn, notably downregulated and approached the level of that in fertilized embryonic stem cells (fESCs). In addition, we acquired a 13.5-day fetus totally derived from GFP-a4 PgESCs with germline contribution by 8-cell under zona pellucida (ZP) injection. In conclusion, four embryos aggregation improves parthenogenetic development, and compensates imprinting genes expression in PgESCs. It implied that a4 PgESCs could serve as a better scientific model applied in translational medicine and imprinting gene study. © 2016 Japanese Society of Developmental Biologists.

The indole compound NC009-1 inhibits aggregation and promotes neurite outgrowth through enhancement of HSPB1 in SCA17 cells and ameliorates the behavioral deficits in SCA17 mice.

PubMed

Chen, Chiung-Mei; Chen, Wan-Ling; Hung, Chen-Ting; Lin, Te-Hsien; Chao, Chih-Ying; Lin, Chih-Hsin; Wu, Yih-Ru; Chang, Kuo-Hsuan; Yao, Ching-Fa; Lee-Chen, Guey-Jen; Su, Ming-Tsan; Hsieh-Li, Hsiu Mei

2018-06-21

Spinocerebellar ataxia type 17 (SCA17) is caused by the expansion of translated CAG repeat in the TATA box binding protein (TBP) gene encoding a long polyglutamine (polyQ) tract in the TBP protein, which leads to intracellular accumulation of aggregated TBP and cell death. The molecular chaperones act in preventing protein aggregation to ameliorate downstream harmful events. In this study, we used Tet-On cells with inducible SCA17 TBP/Q 79 -GFP expression to test five in-house NC009 indole compounds for neuroprotection. We found that both aggregation and polyQ-induced reactive oxygen species can be significantly prohibited by the tested NC009 compounds in Tet-On TBP/Q 79 293 cells. Among the five indole compounds, NC009-1 up-regulated expression of heat shock protein family B (small) member 1 (HSPB1) chaperone to reduce polyQ aggregation and promote neurite outgrowth in neuronal differentiated TBP/Q 79 SH-SY5Y cells. The increased HSPB1 thus ameliorated the increased BH3 interacting domain death agonist (BID), cytochrome c (CYCS) release, and caspase 3 (CASP3) activation which result in apoptosis. Knock down of HSPB1 attenuated the effects of NC009-1 on TBP/Q 79 SH-SY5Y cells, suggesting that HSPB1 might be one of the major pathways involved for NC009-1 effects. NC009-1 further reduced polyQ aggregation in Purkinje cells and ameliorated behavioral deficits in SCA17 TBP/Q 109 transgenic mice. Our results suggest that NC009-1 has a neuroprotective effect on SCA17 cell and mouse models to support its therapeutic potential in SCA17 treatment. Copyright © 2018 Elsevier B.V. All rights reserved.

Three-dimensional printed polymeric system to encapsulate human mesenchymal stem cells differentiated into islet-like insulin-producing aggregates for diabetes treatment

PubMed Central

Sabek, Omaima M; Farina, Marco; Fraga, Daniel W; Afshar, Solmaz; Ballerini, Andrea; Filgueira, Carly S; Thekkedath, Usha R; Grattoni, Alessandro; Gaber, A Osama

2016-01-01

Diabetes is one of the most prevalent, costly, and debilitating diseases in the world. Pancreas and islet transplants have shown success in re-establishing glucose control and reversing diabetic complications. However, both are limited by donor availability, need for continuous immunosuppression, loss of transplanted tissue due to dispersion, and lack of vascularization. To overcome the limitations of poor islet availability, here, we investigate the potential of bone marrow–derived mesenchymal stem cells differentiated into islet-like insulin-producing aggregates. Islet-like insulin-producing aggregates, characterized by gene expression, are shown to be similar to pancreatic islets and display positive immunostaining for insulin and glucagon. To address the limits of current encapsulation systems, we developed a novel three-dimensional printed, scalable, and potentially refillable polymeric construct (nanogland) to support islet-like insulin-producing aggregates’ survival and function in the host body. In vitro studies showed that encapsulated islet-like insulin-producing aggregates maintained viability and function, producing steady levels of insulin for at least 4 weeks. Nanogland—islet-like insulin-producing aggregate technology here investigated as a proof of concept holds potential as an effective and innovative approach for diabetes cell therapy. PMID:27152147

The effects of glutamine/asparagine content on aggregation and heterologous prion induction by yeast prion-like domains.

PubMed

Shattuck, Jenifer E; Waechter, Aubrey C; Ross, Eric D

2017-07-04

Prion-like domains are low complexity, intrinsically disordered domains that compositionally resemble yeast prion domains. Many prion-like domains are involved in the formation of either functional or pathogenic protein aggregates. These aggregates range from highly dynamic liquid droplets to highly ordered detergent-insoluble amyloid-like aggregates. To better understand the amino acid sequence features that promote conversion to stable, detergent-insoluble aggregates, we used the prediction algorithm PAPA to identify predicted aggregation-prone prion-like domains with a range of compositions. While almost all of the predicted aggregation-prone domains formed foci when expressed in cells, the ability to form the detergent-insoluble aggregates was highly correlated with glutamine/asparagine (Q/N) content, suggesting that high Q/N content may specifically promote conversion to the amyloid state in vivo. We then used this data set to examine cross-seeding between prion-like proteins. The prion protein Sup35 requires the presence of a second prion, [PIN + ], to efficiently form prions, but this requirement can be circumvented by the expression of various Q/N-rich protein fragments. Interestingly, almost all of the Q/N-rich domains that formed SDS-insoluble aggregates were able to promote prion formation by Sup35, highlighting the highly promiscuous nature of these interactions.

Curcumin Attenuates Amyloid-β Aggregate Toxicity and Modulates Amyloid-β Aggregation Pathway.

PubMed

Thapa, Arjun; Jett, Stephen D; Chi, Eva Y

2016-01-20

The abnormal misfolding and aggregation of amyloid-β (Aβ) peptides into β-sheet enriched insoluble deposits initiates a cascade of events leading to pathological processes and culminating in cognitive decline in Alzheimer's disease (AD). In particular, soluble oligomeric/prefibrillar Aβ have been shown to be potent neurotoxins. The naturally occurring polyphenol curcumin has been shown to exert a neuroprotective effect against age-related neurodegenerative diseases such as AD. However, its protective mechanism remains unclear. In this study, we investigated the effects of curcumin on the aggregation of Aβ40 as well as Aβ40 aggregate induced neurotoxicity. Our results show that the curcumin does not inhibit Aβ fibril formation, but rather enriches the population of "off-pathway" soluble oligomers and prefibrillar aggregates that were nontoxic. Curcumin also exerted a nonspecific neuroprotective effect, reducing toxicities induced by a range of Aβ conformers, including monomeric, oligomeric, prefibrillar, and fibrillar Aβ. The neuroprotective effect is possibly membrane-mediated, as curcumin reduced the extent of cell membrane permeabilization induced by Aβ aggregates. Taken together, our study shows that curcumin exerts its neuroprotective effect against Aβ induced toxicity through at least two concerted pathways, modifying the Aβ aggregation pathway toward the formation of nontoxic aggregates and ameliorating Aβ-induced toxicity possibly through a nonspecific pathway.

Myxococcus xanthus Developmental Cell Fate Production: Heterogeneous Accumulation of Developmental Regulatory Proteins and Reexamination of the Role of MazF in Developmental Lysis

PubMed Central

Lee, Bongsoo; Holkenbrink, Carina; Treuner-Lange, Anke

2012-01-01

Myxococcus xanthus undergoes a starvation-induced multicellular developmental program during which cells partition into three known fates: (i) aggregation into fruiting bodies followed by differentiation into spores, (ii) lysis, or (iii) differentiation into nonaggregating persister-like cells, termed peripheral rods. As a first step to characterize cell fate segregation, we enumerated total, aggregating, and nonaggregating cells throughout the developmental program. We demonstrate that both cell lysis and cell aggregation begin with similar timing at approximately 24 h after induction of development. Examination of several known regulatory proteins in the separated aggregated and nonaggregated cell fractions revealed previously unknown heterogeneity in the accumulation patterns of proteins involved in type IV pilus (T4P)-mediated motility (PilC and PilA) and regulation of development (MrpC, FruA, and C-signal). As part of our characterization of the cell lysis fate, we set out to investigate the unorthodox MazF-MrpC toxin-antitoxin system which was previously proposed to induce programmed cell death (PCD). We demonstrate that deletion of mazF in two different wild-type M. xanthus laboratory strains does not significantly reduce developmental cell lysis, suggesting that MazF's role in promoting PCD is an adaption to the mutant background strain used previously. PMID:22493014

Role of Different Alpha-Synuclein Strains in Synucleinopathies, Similarities with other Neurodegenerative Diseases

PubMed Central

Melki, Ronald

2015-01-01

Abstract Misfolded protein aggregates are the hallmark of several neurodegenerative diseases in humans. The main protein constituent of these aggregates and the regions within the brain that are affected differ from one neurodegenerative disorder to another. A plethora of reports suggest that distinct diseases have in common the ability of protein aggregates to spread and amplify within the central nervous system. This review summarizes briefly what is known about the nature of the protein aggregates that are infectious and the reason they are toxic to cells. The chameleon property of polypeptides which aggregation into distinct high-molecular weight assemblies is associated to different diseases, in particular, that of alpha-synuclein which aggregation is the hallmark of distinct synucleinopathies, is discussed. Finally, strategies targeting the formation and propagation of structurally distinct alpha-synuclein assemblies associated to different synucleinopathies are presented and their therapeutic and diagnostic potential is discussed. PMID:25757830

Effect of the surfactant Tween 80 on the detachment and dispersal of Salmonella enterica Thompson single cells and aggregates from cilantro leaves as revealed by image analysis

USDA-ARS?s Scientific Manuscript database

Biofilms formed by human enteric pathogens on plants are a great concern to the produce industry. Salmonella enterica has the ability to form biofilms and large aggregates on leaf surfaces, including on cilantro leaves. Aggregates that remained attached after rigorous washing of cilantro leaves and ...

In vitro motility of cells from human epidermoid carcinomas. A study by phase-contrast and reflection-contrast cinematography.

PubMed

Haemmerli, G; Sträuli, P

1981-05-15

The motile behavior of six cell lines derived from human squamous carcinomas (two from the larynx, four from the tongue) was studied by cinematography under phase- and reflection-contrast illumination. The recorded cell activities consist in spreading, stationary and translocation motility, and aggregate formation. Within this common pattern, quantitative modifications ("sub-pattern") are stable properties of the individual cells lines. Such modifications are particularly evident with regard to the dynamic texture of the aggregates which ranges from loose, netlike structures to compact islands with smooth borders. Accordingly, the intensity of cell traffic within and around the aggregates varies considerably. It is discussed to what extent the in vitro motility of the carcinoma cell populations reflects their behavior in the organism and thus the significance of cell movements for invasion.

3D printing human induced pluripotent stem cells with novel hydroxypropyl chitin bioink: scalable expansion and uniform aggregation.

PubMed

Li, Yang; Jiang, Xulin; Li, Ling; Chen, Zhi-Nan; Gao, Ge; Yao, Rui; Sun, Wei

2018-06-28

Human induced pluripotent stem cells (hiPSCs) are more likely to successfully avoid the immunological rejection and ethical problems that are often encountered by human embryonic stem cells in various stem cell studies and applications. To transfer hiPSCs from the laboratory to clinical applications, researchers must obtain sufficient cell numbers. In this study, 3D cell printing was used as a novel method for iPSC scalable expansion. Hydroxypropyl chitin (HPCH), utilized as a new type of bioink, and a set of optimized printing parameters were shown to achieve high cell survival (> 90%) after the printing process and high proliferation efficiency (~ 32.3 folds) during subsequent 10-day culture. After the culture, high levels of pluripotency maintenance were recognized by both qualitative and quantitative detections. Compared with static suspension (SS) culture, hiPSC aggregates formed in 3D printed constructs showed a higher uniformity in size. Using novel dual-fluorescent labelling method, hiPSC aggregates in the constructs were found more inclined to form by <i>in situ</i> proliferation rather than multicellular aggregation. This study revealed unique advantages of non-ionic crosslinking bioink material HPCH, including high gel strength and rapid temperature response in hiPSC printing, and achieved primed state hiPSC printing for the first time. Features achieved in this study, such as high cell yield, high pluripotency maintenance and uniform aggregation provide good foundations for further hiPSC studies on 3D micro-tissue differentiation and drug screening. © 2018 IOP Publishing Ltd.

Inhibitory effect of carotenoids on the degranulation of mast cells via suppression of antigen-induced aggregation of high affinity IgE receptors.

PubMed

Sakai, Shota; Sugawara, Tatsuya; Matsubara, Kiminori; Hirata, Takashi

2009-10-09

Carotenoids have been demonstrated to possess antioxidative and anti-inflammatory effects. However, there is no report that the effects of carotenoids on degranulation of mast cell is critical for type I allergy. In this study, we focused on the effect of carotenoids on antigen-induced degranulation of mast cells. Fucoxanthin, astaxanthin, zeaxanthin, and beta-carotene significantly inhibited the antigen-induced release of beta-hexosaminidase in rat basophilic leukemia 2H3 cells and mouse bone marrow-derived mast cells. Those carotenoids also inhibited antigen-induced aggregation of the high affinity IgE receptor (Fc epsilonRI), which is the most upstream of the degranulating signals of mast cells. Furthermore, carotenoids inhibited Fc epsilonRI-mediated intracellular signaling, such as phosphorylation of Lyn kinase and Fyn kinase. It suggests that the inhibitory effect of carotenoids on the degranulation of mast cells were mainly due to suppressing the aggregation of Fc epsilonRI followed by intracellular signaling. In addition, those carotenoids inhibited antigen-induced translocation of Fc epsilonRI to lipid rafts, which are known as platforms of the aggregation of Fc epsilonRI. We assume that carotenoids may modulate the function of lipid rafts and inhibit the translocation of Fc epsilonRI to lipid rafts. This is the first report that focused on the aggregation of Fc epsilonRI to investigate the mechanism of the inhibitory effects on the degranulation of mast cells and evaluated the functional activity of carotenoids associated with lipid rafts.

Inhibitory Effect of Carotenoids on the Degranulation of Mast Cells via Suppression of Antigen-induced Aggregation of High Affinity IgE Receptors*

PubMed Central

Sakai, Shota; Sugawara, Tatsuya; Matsubara, Kiminori; Hirata, Takashi

2009-01-01

Carotenoids have been demonstrated to possess antioxidative and anti-inflammatory effects. However, there is no report that the effects of carotenoids on degranulation of mast cell is critical for type I allergy. In this study, we focused on the effect of carotenoids on antigen-induced degranulation of mast cells. Fucoxanthin, astaxanthin, zeaxanthin, and β-carotene significantly inhibited the antigen-induced release of β-hexosaminidase in rat basophilic leukemia 2H3 cells and mouse bone marrow-derived mast cells. Those carotenoids also inhibited antigen-induced aggregation of the high affinity IgE receptor (FcϵRI), which is the most upstream of the degranulating signals of mast cells. Furthermore, carotenoids inhibited FcϵRI-mediated intracellular signaling, such as phosphorylation of Lyn kinase and Fyn kinase. It suggests that the inhibitory effect of carotenoids on the degranulation of mast cells were mainly due to suppressing the aggregation of FcϵRI followed by intracellular signaling. In addition, those carotenoids inhibited antigen-induced translocation of FcϵRI to lipid rafts, which are known as platforms of the aggregation of FcϵRI. We assume that carotenoids may modulate the function of lipid rafts and inhibit the translocation of FcϵRI to lipid rafts. This is the first report that focused on the aggregation of FcϵRI to investigate the mechanism of the inhibitory effects on the degranulation of mast cells and evaluated the functional activity of carotenoids associated with lipid rafts. PMID:19700409

Optimising Cell Aggregate Expansion in a Perfused Hollow Fibre Bioreactor via Mathematical Modelling

PubMed Central

Chapman, Lloyd A. C.; Shipley, Rebecca J.; Whiteley, Jonathan P.; Ellis, Marianne J.; Byrne, Helen M.; Waters, Sarah L.

2014-01-01

The need for efficient and controlled expansion of cell populations is paramount in tissue engineering. Hollow fibre bioreactors (HFBs) have the potential to meet this need, but only with improved understanding of how operating conditions and cell seeding strategy affect cell proliferation in the bioreactor. This study is designed to assess the effects of two key operating parameters (the flow rate of culture medium into the fibre lumen and the fluid pressure imposed at the lumen outlet), together with the cell seeding distribution, on cell population growth in a single-fibre HFB. This is achieved using mathematical modelling and numerical methods to simulate the growth of cell aggregates along the outer surface of the fibre in response to the local oxygen concentration and fluid shear stress. The oxygen delivery to the cell aggregates and the fluid shear stress increase as the flow rate and pressure imposed at the lumen outlet are increased. Although the increased oxygen delivery promotes growth, the higher fluid shear stress can lead to cell death. For a given cell type and initial aggregate distribution, the operating parameters that give the most rapid overall growth can be identified from simulations. For example, when aggregates of rat cardiomyocytes that can tolerate shear stresses of up to are evenly distributed along the fibre, the inlet flow rate and outlet pressure that maximise the overall growth rate are predicted to be in the ranges to (equivalent to to ) and to (or 15.6 psi to 15.7 psi) respectively. The combined effects of the seeding distribution and flow on the growth are also investigated and the optimal conditions for growth found to depend on the shear tolerance and oxygen demands of the cells. PMID:25157635

In-silico analysis on biofabricating vascular networks using kinetic Monte Carlo simulations.

PubMed

Sun, Yi; Yang, Xiaofeng; Wang, Qi

2014-03-01

We present a computational modeling approach to study the fusion of multicellular aggregate systems in a novel scaffold-less biofabrication process, known as 'bioprinting'. In this novel technology, live multicellular aggregates are used as fundamental building blocks to make tissues or organs (collectively known as the bio-constructs,) via the layer-by-layer deposition technique or other methods; the printed bio-constructs embedded in maturogens, consisting of nutrient-rich bio-compatible hydrogels, are then placed in bioreactors to undergo the cellular aggregate fusion process to form the desired functional bio-structures. Our approach reported here is an agent-based modeling method, which uses the kinetic Monte Carlo (KMC) algorithm to evolve the cellular system on a lattice. In this method, the cells and the hydrogel media, in which cells are embedded, are coarse-grained to material's points on a three-dimensional (3D) lattice, where the cell-cell and cell-medium interactions are quantified by adhesion and cohesion energies. In a multicellular aggregate system with a fixed number of cells and fixed amount of hydrogel media, where the effect of cell differentiation, proliferation and death are tactically neglected, the interaction energy is primarily dictated by the interfacial energy between cell and cell as well as between cell and medium particles on the lattice, respectively, based on the differential adhesion hypothesis. By using the transition state theory to track the time evolution of the multicellular system while minimizing the interfacial energy, KMC is shown to be an efficient time-dependent simulation tool to study the evolution of the multicellular aggregate system. In this study, numerical experiments are presented to simulate fusion and cell sorting during the biofabrication process of vascular networks, in which the bio-constructs are fabricated via engineering designs. The results predict the feasibility of fabricating the vascular structures via the bioprinting technology and demonstrate the morphological development process during cellular aggregate fusion in various engineering designed structures. The study also reveals that cell sorting will perhaps not significantly impact the final fabricated products, should the maturation process be well-controlled in bioprinting.

Changes in haematology measurements with the Sysmex XT-2000iV during storage of feline blood sampled in EDTA or EDTA plus CTAD.

PubMed

Granat, Fanny; Geffré, Anne; Bourgès-Abella, Nathalie; Braun, Jean-Pierre; Trumel, Catherine

2013-06-01

In veterinary medicine a complete blood cell count (CBC) cannot always be performed within 24 h as usually recommended, particularly for specimens shipped to a reference laboratory. This raises the question of the stability of the variables, especially in ethylenediamine tetra-acetic acid (EDTA) feline blood specimens, known to be prone to in vitro platelet aggregation. Citrate, theophylline, adenosine and dipyridamole (CTAD) has been reported to limit platelet aggregation in feline blood specimens. The aim of this study was to measure the stability of the haematological variables and the platelet aggregation score in EDTA and EDTA plus CTAD (EDCT) feline blood specimens during 48 h of storage at room temperature. Forty-six feline EDTA and EDCT blood specimens were analysed with a Sysmex XT-2000iV analyser, and the platelet count and score of platelet aggregation were estimated immediately and after 24 and 48 h of storage. A significant increase in mean corpuscular volume, haematocrit, reticulocyte and eosinophil counts, and a significant decrease in mean corpuscular haemoglobin concentration and monocyte count were observed. Haemoglobin, mean corpuscular haemoglobin, and red blood cell, white blood cell, neutrophil and lymphocyte counts remained stable. Changes in reticulocyte indexes with time (low fluorescence ratio, medium fluorescence ratio, high fluorescence ratio and immature reticulocyte fraction) were not significant. Changes were generally more pronounced in EDTA than in EDCT. Platelet aggregation decreased markedly in initially highly aggregated EDTA specimens, and increased slightly in initially non- or mildly-aggregated EDTA or EDCT specimens. Platelet counts increased and decreased, or remained stable, respectively. CTAD can reduce storage-induced changes of the haematological variables in feline samples, thus improving the reliability of a CBC and limiting clinical misinterpretations.

LC3 fluorescent puncta in autophagosomes or in protein aggregates can be distinguished by FRAP analysis in living cells

PubMed Central

Wang, Liang; Chen, Min; Yang, Jie; Zhang, Zhihong

2013-01-01

LC3 is a marker protein that is involved in the formation of autophagosomes and autolysosomes, which are usually characterized and monitored by fluorescence microscopy using fluorescent protein-tagged LC3 probes (FP-LC3). FP-LC3 and even endogenous LC3 can also be incorporated into intracellular protein aggregates in an autophagy-independent manner. However, the dynamic process of LC3 associated with autophagosomes and autolysosomes or protein aggregates in living cells remains unclear. Here, we explored the dynamic properties of the two types of FP-LC3-containing puncta using fluorescence microscopy techniques, including fluorescence recovery after photobleaching (FRAP) and fluorescence resonance energy transfer (FRET). The FRAP data revealed that the fluorescent signals of FP-LC3 attached to phagophores or in mature autolysosomes showed either minimal or no recovery after photobleaching, indicating that the dissociation of LC3 from the autophagosome membranes may be very slow. In contrast, FP-LC3 in the protein aggregates exhibited nearly complete recovery (more than 80%) and rapid kinetics of association and dissociation (half-time < 1 sec), indicating a rapid exchange occurs between the aggregates and cytoplasmic pool, which is mainly due to the transient interaction of LC3 and SQSTM1/p62. Based on the distinct dynamic properties of FP-LC3 in the two types of punctate structures, we provide a convenient and useful FRAP approach to distinguish autophagosomes from LC3-involved protein aggregates in living cells. Using this approach, we find the FP-LC3 puncta that adjacently localized to the phagophore marker ATG16L1 were protein aggregate-associated LC3 puncta, which exhibited different kinetics compared with that of autophagic structures. PMID:23482084

The Dictyostelium MAP kinase kinase DdMEK1 regulates chemotaxis and is essential for chemoattractant-mediated activation of guanylyl cyclase.

PubMed Central

Ma, H; Gamper, M; Parent, C; Firtel, R A

1997-01-01

We have identified a MAP kinase kinase (DdMEK1) that is required for proper aggregation in Dictyostelium. Null mutations produce extremely small aggregate sizes, resulting in the formation of slugs and terminal fruiting bodies that are significantly smaller than those of wild-type cells. Time-lapse video microscopy and in vitro assays indicate that the cells are able to produce cAMP waves that move through the aggregation domains. However, these cells are unable to undergo chemotaxis properly during aggregation in response to the chemoattractant cAMP or activate guanylyl cyclase, a known regulator of chemotaxis in Dictyostelium. The activation of guanylyl cyclase in response to osmotic stress is, however, normal. Expression of putative constitutively active forms of DdMEK1 in a ddmek1 null background is capable, at least partially, of complementing the small aggregate size defect and the ability to activate guanylyl cyclase. However, this does not result in constitutive activation of guanylyl cyclase, suggesting that DdMEK1 activity is necessary, but not sufficient, for cAMP activation of guanylyl cyclase. Analysis of a temperature-sensitive DdMEK1 mutant suggests that DdMEK1 activity is required throughout aggregation at the time of guanylyl cyclase activation, but is not essential for proper morphogenesis during the later multicellular stages. The activation of the MAP kinase ERK2, which is essential for chemoattractant activation of adenylyl cyclase, is not affected in ddmek1 null strains, indicating that DdMEK1 does not regulate ERK2 and suggesting that at least two independent MAP kinase cascades control aggregation in Dictyostelium. PMID:9250676

Gap junctions contribute to anchorage-independent clustering of breast cancer cells.

PubMed

Gava, Fabien; Rigal, Lise; Mondesert, Odile; Pesce, Elise; Ducommun, Bernard; Lobjois, Valérie

2018-02-27

Cancer cell aggregation is a key process involved in the formation of clusters of circulating tumor cells. We previously reported that cell-cell adhesion proteins, such as E-cadherin, and desmosomal proteins are involved in cell aggregation to form clusters independently of cell migration or matrix adhesion. Here, we investigated the involvement of gap junction intercellular communication (GJIC) during anchorage-independent clustering of MCF7 breast adenocarcinoma cells. We used live cell image acquisition and analysis to monitor the kinetics of MCF7 cell clustering in the presence/absence of GJIC pharmacological inhibitors and to screen a LOPAC® bioactive compound library. We also used a calcein transfer assay and flow cytometry to evaluate GJIC involvement in cancer cell clustering. We first demonstrated that functional GJIC are established in the early phase of cancer cell aggregation. We then showed that pharmacological inhibition of GJIC using tonabersat and meclofenamate delayed MCF7 cell clustering and reduced calcein transfer. We also found that brefeldin A, an inhibitor of vesicular trafficking, which we identified by screening a small compound library, and latrunculin A, an actin cytoskeleton-disrupting agent, both impaired MCF7 cell clustering and calcein transfer. Our results demonstrate that GJIC are involved from the earliest stages of anchorage-independent cancer cell aggregation. They also give insights into the regulatory mechanisms that could modulate the formation of clusters of circulating tumor cells.

Epothilone D inhibits microglia-mediated spread of alpha-synuclein aggregates.

PubMed

Valdinocci, Dario; Grant, Gary D; Dickson, Tracey C; Pountney, Dean L

2018-04-16

Multiple System Atrophy (MSA) is a progressive neurodegenerative disease characterized by chronic neuroinflammation and widespread α-synuclein (α-syn) cytoplasmic inclusions. Neuroinflammation associated with microglial cells is typically located in brain regions with α-syn deposits. The potential link between microglial cell migration and the transport of pathological α-syn protein in MSA was investigated. Qualitative analysis via immunofluorescence of MSA cases (n = 4) revealed microglial cells bearing α-syn inclusions distal from oligodendrocytes bearing α-syn cytoplasmic inclusions, as well as close interactions between microglia and oligodendrocytes bearing α-syn, suggestive of a potential transfer mechanism between microglia and α-syn bearing cells in MSA and the possibility of microglia acting as a mobile vehicle to spread α-syn between anatomically connected brain regions. Further In vitro experiments using microglial-like differentiated THP-1 cells were conducted to investigate if microglial cells could act as potential transporters of α-syn. Monomeric or aggregated α-syn was immobilized at the centre of glass coverslips and treated with either cell free medium, undifferentiated THP-1 cells or microglial-like phorbol-12-myristate-13-acetate differentiated THP-1 cells (48 h; n = 3). A significant difference in residual immobilized α-syn density was observed between cell free controls and differentiated (p = 0.016) as well as undifferentiated and differentiated THP-1 cells (p = 0.032) when analysed by quantitative immunofluorescence. Furthermore, a significantly greater proportion of differentiated cells were observed bearing α-syn aggregates distal from the immobilized protein than their non-differentiated counterparts (p = 0.025). Similar results were observed with Highly Aggressive Proliferating Immortalised (HAPI) microglial cells, with cells exposed to aggregated α-syn yielding lower residual immobilized α-syn (p = 0.004) and a higher proportion of α-syn positive distal cells (p = 0.001) than cells exposed to monomeric α-syn. Co-treatment of THP-1 groups with the tubulin depolymerisation inhibitor, Epothilone D (EpoD; 10 nM), was conducted to investigate if inhibition of microtubule activity had an effect on cell migration and residual immobilized α-syn density. There was a significant increase in both residual immobilized α-syn between EpoD treated and non-treated differentiated cells exposed to monomeric (p = 0.037) and aggregated (p = 0.018) α-syn, but not with undifferentiated cells. Differentiated THP-1 cells exposed to immobilized aggregated α-syn showed a significant difference in the proportion of distal aggregate bearing cells between EpoD treated and untreated (p = 0.027). The results suggest microglia could play a role in α-syn transport in MSA, a role which could potentially be inhibited therapeutically by EpoD. Copyright © 2018 Elsevier Inc. All rights reserved.

Cell and Particle Interactions and Aggregation During Electrophoretic Motion

NASA Technical Reports Server (NTRS)

Wang, Hua; Zeng, Shulin; Loewenberg, Michael; Todd, Paul; Davis, Robert H.

1996-01-01

The stability and pairwise aggregation rates of small spherical particles under the collective effects of buoyancy-driven motion and electrophoretic migration are analyzed. The particles are assumed to be non-Brownian, with thin double-layers and different zeta potentials. The particle aggregation rates may be enhanced or reduced, respectively, by parallel and antiparallel alignments of the buoyancy-driven and electrophoretic velocities. For antiparallel alignments, with the buoyancy-driven relative velocity exceeding the electrophoretic relative velocity between two widely-separated particles, there is a 'collision-forbidden region' in parameter space due to hydrodynamic interactions; thus, the suspension becomes stable against aggregation.

Biochemical nature of Russell Bodies

PubMed Central

Francesca Mossuto, Maria; Ami, Diletta; Anelli, Tiziana; Fagioli, Claudio; Maria Doglia, Silvia; Sitia, Roberto

2015-01-01

Professional secretory cells produce and release abundant proteins. Particularly in case of mutations and/or insufficient chaperoning, these can aggregate and become toxic within or amongst cells. Immunoglobulins (Ig) are no exception. In the extracellular space, certain Ig-L chains form fibrils causing systemic amyloidosis. On the other hand, Ig variants lacking the first constant domain condense in dilated cisternae of the early secretory compartment, called Russell Bodies (RB), frequently observed in plasma cell dyscrasias, autoimmune diseases and chronic infections. RB biogenesis can be recapitulated in lymphoid and non-lymphoid cells by expressing mutant Ig-μ, providing powerful models to investigate the pathophysiology of endoplasmic reticulum storage disorders. Here we analyze the aggregation propensity and the biochemical features of the intra- and extra-cellular Ig deposits in human cells, revealing β-aggregated features for RB. PMID:26223695

Biochemical nature of Russell Bodies.

PubMed

Mossuto, Maria Francesca; Ami, Diletta; Anelli, Tiziana; Fagioli, Claudio; Doglia, Silvia Maria; Sitia, Roberto

2015-07-30

Professional secretory cells produce and release abundant proteins. Particularly in case of mutations and/or insufficient chaperoning, these can aggregate and become toxic within or amongst cells. Immunoglobulins (Ig) are no exception. In the extracellular space, certain Ig-L chains form fibrils causing systemic amyloidosis. On the other hand, Ig variants lacking the first constant domain condense in dilated cisternae of the early secretory compartment, called Russell Bodies (RB), frequently observed in plasma cell dyscrasias, autoimmune diseases and chronic infections. RB biogenesis can be recapitulated in lymphoid and non-lymphoid cells by expressing mutant Ig-μ, providing powerful models to investigate the pathophysiology of endoplasmic reticulum storage disorders. Here we analyze the aggregation propensity and the biochemical features of the intra- and extra-cellular Ig deposits in human cells, revealing β-aggregated features for RB.

Symbiotic Cell Differentiation and Cooperative Growth in Multicellular Aggregates

PubMed Central

Yamagishi, Jumpei F; Saito, Nen; Kaneko, Kunihiko

2016-01-01

As cells grow and divide under a given environment, they become crowded and resources are limited, as seen in bacterial biofilms and multicellular aggregates. These cells often show strong interactions through exchanging chemicals, as evident in quorum sensing, to achieve mutualism and division of labor. Here, to achieve stable division of labor, three characteristics are required. First, isogenous cells differentiate into several types. Second, this aggregate of distinct cell types shows better growth than that of isolated cells without interaction and differentiation, by achieving division of labor. Third, this cell aggregate is robust with respect to the number distribution of differentiated cell types. Indeed, theoretical studies have thus far considered how such cooperation is achieved when the ability of cell differentiation is presumed. Here, we address how cells acquire the ability of cell differentiation and division of labor simultaneously, which is also connected with the robustness of a cell society. For this purpose, we developed a dynamical-systems model of cells consisting of chemical components with intracellular catalytic reaction dynamics. The reactions convert external nutrients into internal components for cellular growth, and the divided cells interact through chemical diffusion. We found that cells sharing an identical catalytic network spontaneously differentiate via induction from cell-cell interactions, and then achieve division of labor, enabling a higher growth rate than that in the unicellular case. This symbiotic differentiation emerged for a class of reaction networks under the condition of nutrient limitation and strong cell-cell interactions. Then, robustness in the cell type distribution was achieved, while instability of collective growth could emerge even among the cooperative cells when the internal reserves of products were dominant. The present mechanism is simple and general as a natural consequence of interacting cells with limited resources, and is consistent with the observed behaviors and forms of several aggregates of unicellular organisms. PMID:27749898

Physical enviroment of 2-D animal cell aggregates formed in a short pathlength ultrasound standing wave trap.

PubMed

Bazou, Despina; Kuznetsova, Larisa A; Coakley, W Terence

2005-03-01

2-D mammalian cell aggregates can be formed and levitated in a 1.5 MHz single half wavelength ultrasound standing wave trap. The physical environment of cells in such a trap has been examined. Attention was paid to parameters such as temperature, acoustic streaming, cavitation and intercellular forces. The extent to which these factors might be intrusive to a neural cell aggregate levitated in the trap was evaluated. Neural cells were exposed to ultrasound at a pressure amplitude of 0.54 MPa for 30 s; a small aggregate had been formed at the center of the trap. The pressure amplitude was then decreased to 0.27 MPa for 2 min, at which level the aggregation process continued at a slower rate. The pressure amplitude was then decreased to 0.06 MPa for 1 h. Temperature measurements that were conducted in situ with a 200 microm thermocouple over a 30 min period showed that the maximum temperature rise was less than 0.5 K. Acoustic streaming was measured by the particle image velocimetry method (PIV). It was shown that the hydrodynamic stress imposed on cells by acoustic streaming is less than that imposed by gentle preparative centrifugation procedures. Acoustic spectrum analysis showed that cavitation activity does not occur in the cell suspensions sonicated at the above pressures. White noise was detected only at a pressure amplitude of 1.96 MPa. Finally, it was shown that the attractive acoustic force between ultrasonically agglomerated cells is small compared with the normal attractive van der Waals force that operates at close cell surface separations. It is concluded that the standing wave trap operates only to concentrate cells locally, as in tissue, and does not modify the in vitro expression of surface receptor interactions.

Acquisition of Lubrol insolubility, a common step for growth hormone and prolactin in the secretory pathway of neuroendocrine cells.

PubMed

Lee, M S; Zhu, Y L; Chang, J E; Dannies, P S

2001-01-05

Rat prolactin in the dense cores of secretory granules of the pituitary gland is a Lubrol-insoluble aggregate. In GH(4)C(1) cells, newly synthesized rat prolactin and growth hormone were soluble, but after 30 min about 40% converted to a Lubrol-insoluble form. Transport from the endoplasmic reticulum is necessary for conversion to Lubrol insolubility, since incubating cells with brefeldin A or at 15 degrees C reduced formation of insoluble rat (35)S-prolactin. Formation of Lubrol-insoluble aggregates has protein and cell specificity; newly synthesized human growth hormone expressed in AtT20 cells underwent a 40% conversion to Lubrol insolubility with time, but albumin did not, and human growth hormone expressed in COS cells underwent less than 10% conversion to Lubrol insolubility. del32-46 growth hormone, a naturally occurring form of growth hormone, and P89L growth hormone underwent conversion, although they were secreted more slowly, indicating that there is some tolerance in structural requirements for aggregation. An intracellular compartment with an acidic pH is not necessary for conversion to Lubrol insolubility, because incubation with chloroquine or bafilomycin slowed, but did not prevent, the conversion. GH(4)C(1) cells treated with estradiol, insulin, and epidermal growth factor accumulate more secretory granules and store more prolactin, but not more growth hormone, than untreated cells; Lubrol-insoluble aggregates of prolactin and growth hormone formed to the same extent in hormone-treated or untreated GH(4)C(1) cells, but prolactin was retained longer in hormone-treated cells. These findings indicate that aggregation alone is not sufficient to cause retention of secretory granule proteins, and there is an additional selective process.

Macrophage triggering by aggregated immunoglobulins. II. Comparison of IgE and IgG aggregates or immune complexes.

PubMed Central

Pestel, J; Dessaint, J P; Joseph, M; Bazin, H; Capron, A

1984-01-01

Macrophages incubated with complexed or aggregated IgE released beta-glucuronidase (beta-G) within 30 min. In contrast in the presence of aggregated or complexed IgG, macrophages liberated equivalent amount of beta-G only after 6 h incubation. In addition the rapid macrophage stimulation induced by aggregated IgE was also followed by a faster 3H-glucosamine incorporation when compared to the delayed activation caused by aggregated IgG. However, macrophages stimulated either by IgG or by IgE oligomers produced the same percentage of plasminogen activator at 24 h. In contrast, while the interaction between macrophages and aggregated IgE was only followed by a peak of cyclic GMP and a beta-G release during the first 30 min of incubation, the interaction between macrophages and IgG oligomers was accompanied by a simultaneous increase of cyclic GMP and AMP nucleotides and by an absence of beta-G exocytosis. Moreover, the beta-G release induced by aggregated IgE was increased when macrophages were preincubated with aggregated IgG. This additive effect was not observed in the reverse situation. Finally macrophages activated by IgG oligomers were demonstrated to exert a cytotoxic effect on tumour cells and to kill schistosomula in the presence of a low level of complement. Taken together these results underline the peculiar ability of aggregated or complexed IgE to trigger rapidly the macrophage activation compared to aggregated IgG and can explain the important role of complexed IgE in some macrophage dependent cytotoxicity mechanisms (i.e. in parasitic diseases). PMID:6088135

Early outgrowth cells versus endothelial colony forming cells functions in platelet aggregation.

PubMed

Bou Khzam, Lara; Bouchereau, Olivier; Boulahya, Rahma; Hachem, Ahmed; Zaid, Younes; Abou-Saleh, Haissam; Merhi, Yahye

2015-11-09

Endothelial progenitor cells (EPCs) have been implicated in neoangiogenesis, endothelial repair and cell-based therapies for cardiovascular diseases. We have previously shown that the recruitment of EPCs to sites of vascular lesions is facilitated by platelets where EPCs, in turn, modulate platelet function and thrombosis. However, EPCs encompass a heterogeneous population of progenitor cells that may exert different effects on platelet function. Recent evidence suggests the existence of two EPC subtypes: early outgrowth cells (EOCs) and endothelial colony-forming cells (ECFCs). We aimed at characterizing these two EPC subtypes and at identifying their role in platelet aggregation. EOCs and ECFCs were generated from human peripheral blood mononuclear cells (PBMCs) seeded in conditioned media on fibronectin and collagen, respectively. The morphological, phenotypical and functional characteristics of EOCs and ECFCs were assessed by optical and confocal laser scanning microscopes, cell surface markers expression, and Matrigel tube formation. The impact of EOCs and ECFCs on platelet aggregation was monitored in collagen-induced optical aggregometry and compared with PBMCs and human umbilical vein endothelial cells (HUVECs). The levels of the anti-platelet agents' nitric oxide (NO) and prostacyclin (PGI2) released from cultured cells as well as the expression of their respective producing enzymes NO synthases (NOS) and cyclooxygenases (COX) were also assessed. We showed that EOCs display a monocytic-like phenotype whereas ECFCs have an endothelial-like phenotype. We demonstrated that both EOCs and ECFCs and their supernatants inhibited platelet aggregation; however ECFCs were more efficient than EOCs. This could be related to the release of significantly higher amounts of NO and PGI2 from ECFCs, in comparison to EOCs. Indeed, ECFCs, like HUVECs, constitutively express the endothelial (eNOS)-and inducible (iNOS)-NOS isoforms, and COX-1 and weakly express COX-2, whereas EOCs do not constitutively express these NO and PGI2 producing enzymes. The different morphological, phenotypic and more importantly the release of the anti-aggregating agents PGI2 and NO in each EPC subtype are implicated in their respective roles in platelet function and thus, may be linked to the increased efficiency of ECFCs in inhibiting platelet aggregation as compared to EOCs.

Small Molecule Acceptor and Polymer Donor Crystallinity and Aggregation Effects on Microstructure Templating: Understanding Photovoltaic Response in Fullerene-Free Solar Cells

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

Eastham, Nicholas D.; Dudnik, Alexander S.; Aldrich, Thomas J.

Perylenediimide (PDI) small molecule acceptor (SMA) crystallinity and donor polymer aggregation and crystallinity effects on bulk-heterojunction microstructure and polymer solar cell (PSC) performance are systematically investigated. Two highperformance polymers, semicrystalline poly[5-(2-hexyldodecyl)-4Hthieno[3,4-c]pyrrole-4,6(5H)-dione-1,3-yl-alt-4,4''dodecyl-2,2':5',2''- terthiophene-5,5''-diyl] (PTPD3T or D1) and amorphous poly{4,8- bis(5-(2-ethylhexyl)thiophen-2-yl)benzo[1,2-b:4,5-b']dithiophene- 2,6-diyl-alt-(4-(2-ethylhexyl)-3-fluorothieno[3,4-b]thiophene-2-carboxylate-2,6-diyl) (PBDTT-FTTE or D2), are paired with three PDI-based SMAs (A1-A3) of differing crystallinity (A1 is the most, A3 is the least crystalline). The resulting PSC performance trends are strikingly different from those of typical fullerene-based PSCs and are highly material-dependent. The present trends reflect synergistic aggregation propensities between the SMA and polymer components. Importantly, the active layer morphology is templatedmore » by the PDI in some blends and by the polymer in others, with the latter largely governed by the polymer aggregation. Thus, PTPD3T templating capacity increases as self-aggregation increases (greater Mn), optimizing PSC performance with A2, while A3-based cells exhibit an inverse relationship between polymer aggregation and performance, which is dramatically different from fullerene-based PSCs. For PBDTT-FTTE, A2-based cells again deliver the highest PCEs of ~5%, but here both A2 and PBDTT-FTTE (medium Mn) template the morphology. Overall, the present results underscore the importance of nonfullerene acceptor aggregation for optimizing PSC performance and offer guidelines for pairing SMAs with acceptable donor polymers.« less

Stimulation of microtubule-based transport by nucleation of microtubules on pigment granules

PubMed Central

Semenova, Irina; Gupta, Dipika; Usui, Takeo; Hayakawa, Ichiro; Cowan, Ann; Rodionov, Vladimir

2017-01-01

Microtubule (MT)-based transport can be regulated through changes in organization of MT transport tracks, but the mechanisms that regulate these changes are poorly understood. In Xenopus melanophores, aggregation of pigment granules in the cell center involves their capture by the tips of MTs growing toward the cell periphery, and granule aggregation signals facilitate capture by increasing the number of growing MT tips. This increase could be explained by stimulation of MT nucleation either on the centrosome or on the aggregate of pigment granules that gradually forms in the cell center. We blocked movement of pigment granules to the cell center and compared the MT-nucleation activity of the centrosome in the same cells in two signaling states. We found that granule aggregation signals did not stimulate MT nucleation on the centrosome but did increase MT nucleation activity of pigment granules. Elevation of MT-nucleation activity correlated with the recruitment to pigment granules of a major component of MT-nucleation templates, γ-tubulin, and was suppressed by γ-tubulin inhibitors. We conclude that generation of new MT transport tracks by concentration of the leading pigment granules provides a positive feedback loop that enhances delivery of trailing granules to the cell center. PMID:28381426

Interactions of B16F10 melanoma cells aggregated on a cellulose substrate.

PubMed

Hindié, M; Vayssade, M; Dufresne, M; Quéant, S; Warocquier-Clérout, R; Legeay, G; Vigneron, P; Olivier, V; Duval, J-L; Nagel, M-D

2006-09-01

There is evidence that the shape of cells and their contact with a matrix direct the growth and the differentiation of both normal and cancer cells. Cells in 3D culture resemble the in vivo situation more closely than do those in conventional 2D cultures. We have studied the interactions and functions of B16F10 mouse melanoma cells, which spread and grow well on tissue culture polystyrene (tPS), when they were made to aggregate on cellulose-coated Petri dishes (CEL). This aggregation of melanoma cells on CEL was Ca2+ dependent and mediated by N-cadherins. The levels of N-cadherin and beta-catenin transcripts in cells cultured on CEL and tPS were similar, but those on CEL contained less beta-catenin protein. Immunoprecipitation and immunostaining showed that both N-cadherins and beta-catenins were present at the membranes of cells on CEL. Cells proliferated significantly more slowly after 48 h on CEL and the cellulose coating caused most of them to arrest in G1. We also compared the melanin contents and tyrosinase activity of cells on CEL and controls grown on tPS. Melanogenesis was induced in cells aggregated on CEL. A cellulose substrate thus appears to be an outstanding tool for studying cell-cell interactions and cell functions in 3D cultures.

An Enzyme from Aristolochia indica Destabilizes Fibrin-β Amyloid Co-Aggregate: Implication in Cerebrovascular Diseases

PubMed Central

Bhattacharjee, Payel; Bhattacharyya, Debasish

2015-01-01

Fibrinogen and β-amyloid (Aβ) peptide independently form ordered aggregates but in combination, they form disordered structures which are resistant to fibrinolytic enzymes like plasmin and cause severity in cerebral amyloid angiopathy (CAA). A novel enzyme of 31.3 kDa has been isolated from the root of the medicinal plant Aristolochia indica that showed fibrinolytic as well as fibrin-Aβ co-aggregate destabilizing properties. This enzyme is functionally distinct from plasmin. Thrombolytic action of the enzyme was demonstrated in rat model. The potency of the plant enzyme in degrading fibrin and fibrin-plasma protein (Aβ, human serum albumin, lysozyme, transthyretin and fibronectin) co-aggregates was demonstrated by atomic force microscopy, scanning electron microscopy and confocal microscopy that showed better potency of the plant enzyme as compared to plasmin. Moreover, the plant enzyme inhibited localization of the co-aggregate inside SH-SY5Y human neuroblastoma cells and also co-aggregate induced cytotoxicity. Plasmin was inefficient in this respect. In the background of limited options for fragmentation of these co-aggregates, the plant enzyme may appear as a potential proteolytic enzyme. PMID:26545113

Cryptic Amyloidogenic Elements in the 3′ UTRs of Neurofilament Genes Trigger Axonal Neuropathy

PubMed Central

Rebelo, Adriana P.; Abrams, Alexander J.; Cottenie, Ellen; Horga, Alejandro; Gonzalez, Michael; Bis, Dana M.; Sanchez-Mejias, Avencia; Pinto, Milena; Buglo, Elena; Markel, Kasey; Prince, Jeffrey; Laura, Matilde; Houlden, Henry; Blake, Julian; Woodward, Cathy; Sweeney, Mary G.; Holton, Janice L.; Hanna, Michael; Dallman, Julia E.; Auer-Grumbach, Michaela; Reilly, Mary M.; Zuchner, Stephan

2016-01-01

Abnormal protein aggregation is observed in an expanding number of neurodegenerative diseases. Here, we describe a mechanism for intracellular toxic protein aggregation induced by an unusual mutation event in families affected by axonal neuropathy. These families carry distinct frameshift variants in NEFH (neurofilament heavy), leading to a loss of the terminating codon and translation of the 3′ UTR into an extra 40 amino acids. In silico aggregation prediction suggested the terminal 20 residues of the altered NEFH to be amyloidogenic, which we confirmed experimentally by serial deletion analysis. The presence of this amyloidogenic motif fused to NEFH caused prominent and toxic protein aggregates in transfected cells and disrupted motor neurons in zebrafish. We identified a similar aggregation-inducing mechanism in NEFL (neurofilament light) and FUS (fused in sarcoma), in which mutations are known to cause aggregation in Charcot-Marie-Tooth disease and amyotrophic lateral sclerosis, respectively. In summary, we present a protein-aggregation-triggering mechanism that should be taken into consideration during the evaluation of stop-loss variants. PMID:27040688

Rationally Designed Peptidomimetic Modulators of Aβ Toxicity in Alzheimer's Disease

NASA Astrophysics Data System (ADS)

Rajasekhar, K.; Suresh, S. N.; Manjithaya, Ravi; Govindaraju, T.

2015-01-01

Alzheimer's disease is one of the devastating illnesses mankind is facing in the 21st century. The main pathogenic event in Alzheimer's disease is believed to be the aggregation of the β-amyloid (Aβ) peptides into toxic aggregates. Molecules that interfere with this process may act as therapeutic agents for the treatment of the disease. Use of recognition unit based peptidomimetics as inhibitors are a promising approach, as they exhibit greater protease stability compared to natural peptides. Here, we present peptidomimetic inhibitors of Aβ aggregation designed based on the KLVFF (P1) sequence that is known to bind Aβ aggregates. We improved inhibition efficiency of P1 by introducing multiple hydrogen bond donor-acceptor moieties (thymine/barbiturate) at the N-terminal (P2 and P3), and blood serum stability by modifying the backbone by incorporating sarcosine (N-methylglycine) units at alternate positions (P4 and P5). The peptidomimetics showed moderate to good activity in both inhibition and dissolution of Aβ aggregates as depicted by thioflavin assay, circular dichroism (CD) measurements and microscopy (TEM). The activity of P4 and P5 were studied in a yeast cell model showing Aβ toxicity. P4 and P5 could rescue yeast cells from Aβ toxicity and Aβ aggregates were cleared by the process of autophagy.

Metabolic states following accumulation of intracellular aggregates: implications for neurodegenerative diseases.

PubMed

Vazquez, Alexei

2013-01-01

The formation of intracellular aggregates is a common etiology of several neurodegenerative diseases. Mitochondrial defects and oxidative stress has been pointed as the major mechanistic links between the accumulation of intracellular aggregates and cell death. In this work we propose a "metabolic cell death by overcrowding" as an alternative hypothesis. Using a model of neuron metabolism, we predict that as the concentration of protein aggregates increases the neurons transit through three different metabolic phases. The first phase (0-6 mM) corresponds with the normal neuron state, where the neuronal activity is sustained by the oxidative phosphorylation of lactate. The second phase (6-8.6 mM) is characterized by a mixed utilization of lactate and glucose as energy substrates and a switch from ammonia uptake to ammonia release by neurons. In the third phase (8.6-9.3 mM) neurons are predicted to support their energy demands from glycolysis and an alternative pathway for energy generation, involving reactions from serine synthesis, one carbon metabolism and the glycine cleavage system. The model also predicts a decrease in the maximum neuronal capacity for energy generation with increasing the concentration of protein aggregates. Ultimately this maximum capacity becomes zero when the protein aggregates reach a concentration of about 9.3 mM, predicting the cessation of neuronal activity.

Protein aggregation induced during glass bead lysis of yeast

PubMed Central

Papanayotou, Irene; Sun, Beimeng; Roth, Amy F.; Davis, Nicholas G.

2013-01-01

Yeast cell lysates produced by mechanical glass bead disruption are widely used in a variety of applications, including for the analysis of native function, e.g. protein–protein interaction, enzyme assays and membrane fractionations. Below, we report a striking case of protein denaturation and aggregation that is induced by this lysis protocol. Most of this analysis focuses on the type 1 casein kinase Yck2, which normally tethers to the plasma membrane through C-terminal palmitoylation. Surprisingly, when cells are subjected to glass bead disruption, non-palmitoylated, cytosolic forms of the kinase denature and aggregate, while membrane-associated forms, whether attached through their native palmitoyl tethers or through a variety of artificial membrane-tethering sequences, are wholly protected from denaturation and aggregation. A wider look at the yeast proteome finds that, while the majority of proteins resist glass bead-induced aggregation, a significant subset does, in fact, succumb to such denaturation. Thus, yeast researchers should be aware of this potential artifact when embarking on biochemical analyses that employ glass bead lysates to look at native protein function. Finally, we demonstrate an experimental utility for glass bead-induced aggregation, using its fine discrimination of membrane-associated from non-associated Yck2 forms to discern fractional palmitoylation states of Yck2 mutants that are partially defective for palmitoylation. PMID:20641011

A microfabricated platform to form three-dimensional toroidal multicellular aggregate.

PubMed

Masuda, Taisuke; Takei, Natsuki; Nakano, Takuma; Anada, Takahisa; Suzuki, Osamu; Arai, Fumihito

2012-12-01

Techniques that allow cells to self-assemble into three-dimensional (3D) spheroid microtissues provide powerful in vitro models that are becoming increasingly popular in fields such as stem cell research, tissue engineering, and cancer biology. Appropriate simulation of the 3D environment in which tissues normally develop and function is crucial for the engineering of in vitro models that can be used for the formation of complex tissues. We have developed a unique multicellular aggregate formation platform that utilizes a maskless gray-scale photolithography. The cellular aggregate formed using this platform has a toroidal-like geometry and includes a micro lumen that facilitates the supply of oxygen and growth factors and the expulsion of waste products. As a result, this platform was capable of rapidly producing hundreds of multicellular aggregates at a time, and of regulating the diameter of aggregates with complex design. These toroidal multicellular aggregates can grow as long-term culture. In addition, the micro lumen can be used as a continuous channel and for the insertion of a vascular system or a nerve system into the assembled tissue. These platform characteristics highlight its potential to be used in a wide variety of applications, e.g. as a bioactuator, as a micro-machine component or in drug screening and tissue engineering.

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

Landreh, Michael; Stukenborg, Jan-Bernd; Willander, Hanna

Highlights: Black-Right-Pointing-Pointer Insulin and C-peptide can interact under insulin fibril forming conditions. Black-Right-Pointing-Pointer C-peptide is incorporated into insulin aggregates and alters aggregation lag time. Black-Right-Pointing-Pointer C-peptide changes insulin fibril morphology and affects backbone accessibility. Black-Right-Pointing-Pointer C-peptide may be a regulator of fibril formation by {beta}-cell granule proteins. -- Abstract: Insulin aggregation can prevent rapid insulin uptake and cause localized amyloidosis in the treatment of type-1 diabetes. In this study, we investigated the effect of C-peptide, the 31-residue peptide cleaved from proinsulin, on insulin fibrillation at optimal conditions for fibrillation. This is at low pH and high concentration, when the fibrilsmore » formed are regular and extended. We report that C-peptide then modulates the insulin aggregation lag time and profoundly changes the fibril appearance, to rounded clumps of short fibrils, which, however, still are Thioflavine T-positive. Electrospray ionization mass spectrometry also indicates that C-peptide interacts with aggregating insulin and is incorporated into the aggregates. Hydrogen/deuterium exchange mass spectrometry further reveals reduced backbone accessibility in insulin aggregates formed in the presence of C-peptide. Combined, these effects are similar to those of C-peptide on islet amyloid polypeptide fibrillation and suggest that C-peptide has a general ability to interact with amyloidogenic proteins from pancreatic {beta}-cell granules. Considering the concentrations, these peptide interactions should be relevant also during physiological secretion, and even so at special sites post-secretory or under insulin treatment conditions in vivo.« less

The Effect of Pulsatile Versus Nonpulsatile Blood Flow on Viscoelasticity and Red Blood Cell Aggregation in Extracorporeal Circulation

PubMed Central

Ahn, Chi Bum; Kang, Yang Jun; Kim, Myoung Gon; Yang, Sung; Lim, Choon Hak; Son, Ho Sung; Kim, Ji Sung; Lee, So Young; Son, Kuk Hui; Sun, Kyung

2016-01-01

Background Extracorporeal circulation (ECC) can induce alterations in blood viscoelasticity and cause red blood cell (RBC) aggregation. In this study, the authors evaluated the effects of pump flow pulsatility on blood viscoelasticity and RBC aggregation. Methods Mongrel dogs were randomly assigned to two groups: a nonpulsatile pump group (n=6) or a pulsatile pump group (n=6). After ECC was started at a pump flow rate of 80 mL/kg/min, cardiac fibrillation was induced. Blood sampling was performed before and at 1, 2, and 3 hours after ECC commencement. To eliminate bias induced by hematocrit and plasma, all blood samples were adjusted to a hematocrit of 45% using baseline plasma. Blood viscoelasticity, plasma viscosity, hematocrit, arterial blood gas analysis, central venous O2 saturation, and lactate were measured. Results The blood viscosity and aggregation index decreased abruptly 1 hour after ECC and then remained low during ECC in both groups, but blood elasticity did not change during ECC. Blood viscosity, blood elasticity, plasma viscosity, and the aggregation index were not significantly different in the groups at any time. Hematocrit decreased abruptly 1 hour after ECC in both groups due to dilution by the priming solution used. Conclusion After ECC, blood viscoelasticity and RBC aggregation were not different in the pulsatile and nonpulsatile groups in the adult dog model. Furthermore, pulsatile flow did not have a more harmful effect on blood viscoelasticity or RBC aggregation than nonpulsatile flow. PMID:27298790

The Effect of Pulsatile Versus Nonpulsatile Blood Flow on Viscoelasticity and Red Blood Cell Aggregation in Extracorporeal Circulation.

PubMed

Ahn, Chi Bum; Kang, Yang Jun; Kim, Myoung Gon; Yang, Sung; Lim, Choon Hak; Son, Ho Sung; Kim, Ji Sung; Lee, So Young; Son, Kuk Hui; Sun, Kyung

2016-06-01

Extracorporeal circulation (ECC) can induce alterations in blood viscoelasticity and cause red blood cell (RBC) aggregation. In this study, the authors evaluated the effects of pump flow pulsatility on blood viscoelasticity and RBC aggregation. Mongrel dogs were randomly assigned to two groups: a nonpulsatile pump group (n=6) or a pulsatile pump group (n=6). After ECC was started at a pump flow rate of 80 mL/kg/min, cardiac fibrillation was induced. Blood sampling was performed before and at 1, 2, and 3 hours after ECC commencement. To eliminate bias induced by hematocrit and plasma, all blood samples were adjusted to a hematocrit of 45% using baseline plasma. Blood viscoelasticity, plasma viscosity, hematocrit, arterial blood gas analysis, central venous O2 saturation, and lactate were measured. The blood viscosity and aggregation index decreased abruptly 1 hour after ECC and then remained low during ECC in both groups, but blood elasticity did not change during ECC. Blood viscosity, blood elasticity, plasma viscosity, and the aggregation index were not significantly different in the groups at any time. Hematocrit decreased abruptly 1 hour after ECC in both groups due to dilution by the priming solution used. After ECC, blood viscoelasticity and RBC aggregation were not different in the pulsatile and nonpulsatile groups in the adult dog model. Furthermore, pulsatile flow did not have a more harmful effect on blood viscoelasticity or RBC aggregation than nonpulsatile flow.

ActA Promotes Listeria monocytogenes Aggregation, Intestinal Colonization and Carriage

PubMed Central

Travier, Laetitia; Guadagnini, Stéphanie; Gouin, Edith; Dufour, Alexandre; Chenal-Francisque, Viviane; Cossart, Pascale; Olivo-Marin, Jean-Christophe; Ghigo, Jean-Marc; Disson, Olivier; Lecuit, Marc

2013-01-01

Listeria monocytogenes (Lm) is a ubiquitous bacterium able to survive and thrive within the environment and readily colonizes a wide range of substrates, often as a biofilm. It is also a facultative intracellular pathogen, which actively invades diverse hosts and induces listeriosis. So far, these two complementary facets of Lm biology have been studied independently. Here we demonstrate that the major Lm virulence determinant ActA, a PrfA-regulated gene product enabling actin polymerization and thereby promoting its intracellular motility and cell-to-cell spread, is critical for bacterial aggregation and biofilm formation. We show that ActA mediates Lm aggregation via direct ActA-ActA interactions and that the ActA C-terminal region, which is not involved in actin polymerization, is essential for aggregation in vitro. In mice permissive to orally-acquired listeriosis, ActA-mediated Lm aggregation is not observed in infected tissues but occurs in the gut lumen. Strikingly, ActA-dependent aggregating bacteria exhibit an increased ability to persist within the cecum and colon lumen of mice, and are shed in the feces three order of magnitude more efficiently and for twice as long than bacteria unable to aggregate. In conclusion, this study identifies a novel function for ActA and illustrates that in addition to contributing to its dissemination within the host, ActA plays a key role in Lm persistence within the host and in transmission from the host back to the environment. PMID:23382675

Expansion of 3D human induced pluripotent stem cell aggregates in bioreactors: Bioprocess intensification and scaling-up approaches.

PubMed

Abecasis, Bernardo; Aguiar, Tiago; Arnault, Émilie; Costa, Rita; Gomes-Alves, Patricia; Aspegren, Anders; Serra, Margarida; Alves, Paula M

2017-03-20

Human induced pluripotent stem cells (hiPSC) are attractive tools for drug screening and disease modeling and promising candidates for cell therapy applications. However, to achieve the high numbers of cells required for these purposes, scalable and clinical-grade technologies must be established. In this study, we use environmentally controlled stirred-tank bioreactors operating in perfusion as a powerful tool for bioprocess intensification of hiPSC production. We demonstrate the importance of controlling the dissolved oxygen concentration at low levels (4%) and perfusion at 1.3day -1 dilution rate to improve hiPSC growth as aggregates in a xeno-free medium. This strategy allowed for increased cell specific growth rate, maximum volumetric concentrations (4.7×10 6 cell/mL) and expansion factors (approximately 19 in total cells), resulting in a 2.6-fold overall improvement in cell yields. Extensive cell characterization, including whole proteomic analysis, was performed to confirm that cells' pluripotent phenotype was maintained during culture. A scalable protocol for continuous expansion of hiPSC aggregates in bioreactors was implemented using mechanical dissociation for aggregate disruption and cell passaging. A total expansion factor of 1100 in viable cells was obtained in 11days of culture, while cells maintained their proliferation capacity, pluripotent phenotype and potential as well as genomic stability after 3 sequential passages in bioreactors. Copyright © 2017 Elsevier B.V. All rights reserved.

Organoids with cancer stem cell-like properties secrete exosomes and HSP90 in a 3D nanoenvironment.

PubMed

Eguchi, Takanori; Sogawa, Chiharu; Okusha, Yuka; Uchibe, Kenta; Iinuma, Ryosuke; Ono, Kisho; Nakano, Keisuke; Murakami, Jun; Itoh, Manabu; Arai, Kazuya; Fujiwara, Toshifumi; Namba, Yuri; Murata, Yoshiki; Ohyama, Kazumi; Shimomura, Manami; Okamura, Hirohiko; Takigawa, Masaharu; Nakatsura, Tetsuya; Kozaki, Ken-Ichi; Okamoto, Kuniaki; Calderwood, Stuart K

2018-01-01

Ability to form cellular aggregations such as tumorspheres and spheroids have been used as a morphological marker of malignant cancer cells and in particular cancer stem cells (CSC). However, the common definition of the types of cellular aggregation formed by cancer cells has not been available. We examined morphologies of 67 cell lines cultured on three dimensional morphology enhancing NanoCulture Plates (NCP) and classified the types of cellular aggregates that form. Among the 67 cell lines, 49 cell lines formed spheres or spheroids, 8 cell lines formed grape-like aggregation (GLA), 8 cell lines formed other types of aggregation, and 3 cell lines formed monolayer sheets. Seven GLA-forming cell lines were derived from adenocarcinoma among the 8 lines. A neuroendocrine adenocarcinoma cell line PC-3 formed asymmetric GLA with ductal structures on the NCPs and rapidly growing asymmetric tumors that metastasized to lymph nodes in immunocompromised mice. In contrast, another adenocarcinoma cell line DU-145 formed spheroids in vitro and spheroid-like tumors in vivo that did not metastasize to lymph nodes until day 50 after transplantation. Culture in the 3D nanoenvironment and in a defined stem cell medium enabled the neuroendocrine adenocarcinoma cells to form slowly growing large organoids that expressed multiple stem cell markers, neuroendocrine markers, intercellular adhesion molecules, and oncogenes in vitro. In contrast, the more commonly used 2D serum-contained environment reduced intercellular adhesion and induced mesenchymal transition and promoted rapid growth of the cells. In addition, the 3D stemness nanoenvironment promoted secretion of HSP90 and EpCAM-exosomes, a marker of CSC phenotype, from the neuroendocrine organoids. These findings indicate that the NCP-based 3D environment enables cells to form stem cell tumoroids with multipotency and model more accurately the in vivo tumor status at the levels of morphology and gene expression.

Organoids with cancer stem cell-like properties secrete exosomes and HSP90 in a 3D nanoenvironment

PubMed Central

Okusha, Yuka; Uchibe, Kenta; Iinuma, Ryosuke; Ono, Kisho; Nakano, Keisuke; Murakami, Jun; Itoh, Manabu; Arai, Kazuya; Fujiwara, Toshifumi; Namba, Yuri; Murata, Yoshiki; Ohyama, Kazumi; Shimomura, Manami; Okamura, Hirohiko; Takigawa, Masaharu; Nakatsura, Tetsuya; Kozaki, Ken-ichi; Okamoto, Kuniaki; Calderwood, Stuart K.

2018-01-01

Ability to form cellular aggregations such as tumorspheres and spheroids have been used as a morphological marker of malignant cancer cells and in particular cancer stem cells (CSC). However, the common definition of the types of cellular aggregation formed by cancer cells has not been available. We examined morphologies of 67 cell lines cultured on three dimensional morphology enhancing NanoCulture Plates (NCP) and classified the types of cellular aggregates that form. Among the 67 cell lines, 49 cell lines formed spheres or spheroids, 8 cell lines formed grape-like aggregation (GLA), 8 cell lines formed other types of aggregation, and 3 cell lines formed monolayer sheets. Seven GLA-forming cell lines were derived from adenocarcinoma among the 8 lines. A neuroendocrine adenocarcinoma cell line PC-3 formed asymmetric GLA with ductal structures on the NCPs and rapidly growing asymmetric tumors that metastasized to lymph nodes in immunocompromised mice. In contrast, another adenocarcinoma cell line DU-145 formed spheroids in vitro and spheroid-like tumors in vivo that did not metastasize to lymph nodes until day 50 after transplantation. Culture in the 3D nanoenvironment and in a defined stem cell medium enabled the neuroendocrine adenocarcinoma cells to form slowly growing large organoids that expressed multiple stem cell markers, neuroendocrine markers, intercellular adhesion molecules, and oncogenes in vitro. In contrast, the more commonly used 2D serum-contained environment reduced intercellular adhesion and induced mesenchymal transition and promoted rapid growth of the cells. In addition, the 3D stemness nanoenvironment promoted secretion of HSP90 and EpCAM-exosomes, a marker of CSC phenotype, from the neuroendocrine organoids. These findings indicate that the NCP-based 3D environment enables cells to form stem cell tumoroids with multipotency and model more accurately the in vivo tumor status at the levels of morphology and gene expression. PMID:29415026

Thermodynamic evidence for Ca2+-mediated self-aggregation of Lewis X gold glyconanoparticles. A model for cell adhesion via carbohydrate-carbohydrate interaction.

PubMed

de la Fuente, Jesús M; Eaton, Peter; Barrientos, Africa G; Menéndez, Margarita; Penadés, Soledad

2005-05-04

Thermodynamic evidence for the selective Ca(2+)-mediated self-aggregation via carbohydrate-carbohydrate interactions of gold glyconanoparticles functionalized with the disaccharides lactose (lacto-Au) and maltose (malto-Au), or the biologically relevant trisaccharide Lewis X (Le(X)-Au), was obtained by isothermal titration calorimetry. The aggregation process was also directly visualized by atomic force microscopy. It was shown in the case of the trisaccharide Lewis X that the Ca(2+)-mediated aggregation is a slow process that takes place with a decrease in enthalpy of 160 +/- 30 kcal mol(-)(1), while the heat evolved in the case of lactose and maltose glyconanoparticles was very low and thermal equilibrium was quickly achieved. Measurements in the presence of Mg(2+) and Na(+) cations confirm the selectivity for Ca(2+) of Le(X)-Au glyconanoparticles. The relevance of this result to cell-cell adhesion process mediated by carbohydrate-carbohydrate interactions is discussed.

Combined in Vitro Cell-Based/in Silico Screening of Naturally Occurring Flavonoids and Phenolic Compounds as Potential Anti-Alzheimer Drugs.

PubMed

Espargaró, Alba; Ginex, Tiziana; Vadell, Maria Del Mar; Busquets, Maria A; Estelrich, Joan; Muñoz-Torrero, Diego; Luque, F Javier; Sabate, Raimon

2017-02-24

Alzheimer's disease (AD) is the main cause of dementia in people over 65 years. One of the major culprits in AD is the self-aggregation of amyloid-β peptide (Aβ), which has stimulated the search for small molecules able to inhibit Aβ aggregation. In this context, we recently reported a simple, but effective in vitro cell-based assay to evaluate the potential antiaggregation activity of putative Aβ aggregation inhibitors. In this work this assay was used together with docking and molecular dynamics simulations to analyze the anti-Aβ aggregation activity of several naturally occurring flavonoids and phenolic compounds. The results showed that rosmarinic acid, melatonin, and o-vanillin displayed zero or low inhibitory capacity, curcumin was found to have an intermediate inhibitory potency, and apigenin and quercetin showed potent antiaggregation activity. Finally, the suitability of the combined in vitro cell-based/in silico approach to distinguish between active and inactive compounds was further assessed for an additional set of flavonols and dihydroflavonols.

Method for Producing Non-Neoplastic, Three Dimensional, Mammalian Tissue and Cell Aggregates Under Microgravity Culture Conditions and the Products Produced Therefrom

NASA Technical Reports Server (NTRS)

Goodwin, Thomas J. (Inventor); Wolf, David A. (Inventor); Spaulding, Glenn F. (Inventor); Prewett, Tracey L. (Inventor)

1996-01-01

Normal mammalian tissue and the culturing process has been developed for the three groups of organ, structural, and blood tissue. The cells are grown in vitro under microgravity culture conditions and form three dimensional cells aggregates with normal cell function. The microgravity culture conditions may be microgravity or simulated microgravity created in a horizontal rotating wall culture vessel.

In vitro motility evaluation of aggregated cancer cells by means of automatic image processing.

PubMed

De Hauwer, C; Darro, F; Camby, I; Kiss, R; Van Ham, P; Decaesteker, C

1999-05-01

Set up of an automatic image processing based method that enables the motility of in vitro aggregated cells to be evaluated for a number of hours. Our biological model included the PC-3 human prostate cancer cell line growing as a monolayer on the bottom of Falcon plastic dishes containing conventional culture media. Our equipment consisted of an incubator, an inverted phase contrast microscope, a Charge Coupled Device (CCD) video camera, and a computer equipped with an image processing software developed in our laboratory. This computer-assisted microscope analysis of aggregated cells enables global cluster motility to be evaluated. This analysis also enables the trajectory of each cell to be isolated and parametrized within a given cluster or, indeed, the trajectories of individual cells outside a cluster. The results show that motility inside a PC-3 cluster is not restricted to slight motion due to cluster expansion, but rather consists of a marked cell movement within the cluster. The proposed equipment enables in vitro aggregated cell motility to be studied. This method can, therefore, be used in pharmacological studies in order to select anti-motility related compounds. The compounds selected by the equipment described could then be tested in vivo as potential anti-metastatic.

Aggregate Formation of Oligonucleotides that Assist Molecular Imaging for Tracking of the Oxygen Status in Tumor Tissue.

PubMed

Yoshihara, Kazuki; Takagi, Kohei; Son, Aoi; Kurihara, Ryohsuke; Tanabe, Kazuhito

2017-08-17

The use of DNA aggregates could be a promising strategy for the molecular imaging of biological functions. Herein, phosphorescent oligodeoxynucleotides were designed with the aim of visualizing oxygen fluctuation in tumor cells. DNA-ruthenium conjugates (DRCs) that consisted of oligodeoxynucleotides, a phosphorescent ruthenium complex, a pyrene unit for high oxygen responsiveness, and a nitroimidazole unit as a tumor-targeting unit were prepared. In general, oligonucleotides have low cell permeability because of their own negative charges; however, the DRC formed aggregates in aqueous solution due to the hydrophobic pyrene and nitroimidazole groups, and smoothly penetrated the cellular membrane to accumulate in tumor cells in a hypoxia-selective manner. The oxygen-dependent phosphorescence of DRC in cells was also observed. In vivo experiments revealed that aggregates of DRC accumulated in hypoxic tumor tissue that was transplanted into the left leg of mice, and showed that oxygen fluctuations in tumor tissue could be monitored by tracking of the phosphorescence emission of DRC. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Solvents induced ZnO nanoparticles aggregation associated with their interfacial effect on organic solar cells.

PubMed

Li, Pandeng; Jiu, Tonggang; Tang, Gang; Wang, Guojie; Li, Jun; Li, Xiaofang; Fang, Junfeng

2014-10-22

ZnO nanofilm as a cathode buffer layer has surface defects due to the aggregations of ZnO nanoparticles, leading to poor device performance of organic solar cells. In this paper, we report the ZnO nanoparticles aggregations in solution can be controlled by adjusting the solvents ratios (chloroform vs methanol). These aggregations could influence the morphology of ZnO film. Therefore, compact and homogeneous ZnO film can be obtained to help achieve a preferable power conversion efficiency of 8.54% in inverted organic solar cells. This improvement is attributed to the decreased leakage current and the increased electron-collecting efficiency as well as the improved interface contact with the active layer. In addition, we find the enhanced maximum exciton generation rate and exciton dissociation probability lead to the improvement of device performance due to the preferable ZnO dispersion. Compared to other methods of ZnO nanofilm fabrication, it is the more convenient, moderate, and effective to get a preferable ZnO buffer layer for high-efficiency organic solar cells.

Pigment granule translocation in red ovarian chromatophores from the palaemonid shrimp Macrobrachium olfersi (Weigmann, 1836): functional roles for the cytoskeleton and its molecular motors.

PubMed

Milograna, Sarah Ribeiro; Ribeiro, Márcia Regina; Baqui, Munira Muhammad Abdel; McNamara, John Campbell

2014-12-01

The binding of red pigment concentrating hormone (RPCH) to membrane receptors in crustacean chromatophores triggers Ca²⁺/cGMP signaling cascades that activate cytoskeletal motors, driving pigment granule translocation. We investigate the distributions of microfilaments and microtubules and their associated molecular motors, myosin and dynein, by confocal and transmission electron microscopy, evaluating a functional role for the cytoskeleton in pigment translocation using inhibitors of polymer turnover and motor activity in vitro. Microtubules occupy the chromatophore cell extensions whether the pigment granules are aggregated or dispersed. The inhibition of microtubule turnover by taxol induces pigment aggregation and inhibits re-dispersion. Phalloidin-FITC actin labeling, together with tannic acid fixation and ultrastructural analysis, reveals that microfilaments form networks associated with the pigment granules. Actin polymerization induced by jasplaquinolide strongly inhibits RPCH-induced aggregation, causes spontaneous pigment dispersion, and inhibits pigment re-dispersion. Inhibition of actin polymerization by latrunculin-A completely impedes pigment aggregation and re-dispersion. Confocal immunocytochemistry shows that non-muscle myosin II (NMMII) co-localizes mainly with pigment granules while blebbistatin inhibition of NMMII strongly reduces the RPCH response, also inducing spontaneous pigment dispersion. Myosin II and dynein also co-localize with the pigment granules. Inhibition of dynein ATPase by erythro-9-(2-hydroxy-3-nonyl) adenine induces aggregation, inhibits RPCH-triggered aggregation, and inhibits re-dispersion. Granule aggregation and dispersion depend mainly on microfilament integrity although microtubules may be involved. Both cytoskeletal polymers are functional only when subunit turnover is active. Myosin and dynein may be the molecular motors that drive pigment aggregation. These mechanisms of granule translocation in crustacean chromatophores share various features with those of vertebrate pigment cells. Copyright © 2014 Elsevier Inc. All rights reserved.

Deciphering Structural Intermediates and Genotoxic Fibrillar Aggregates of Albumins: A Molecular Mechanism Underlying for Degenerative Diseases

PubMed Central

Naeem, Aabgeena; Amani, Samreen

2013-01-01

The misfolding and aggregation of proteins is involved in some of the most prevalent neurodegenerative disorders. The importance of human serum albumin (HSA) stems from the fact that it is involved in bio-regulatory and transport phenomena. Here the effect of acetonitrile (ACN) on the conformational stability of HSA and by comparison, ovalbumin (OVA) has been evaluated in the presence and absence of NaCl. The results show the presence of significant amount of secondary structure in HSA at 70% ACN and in OVA at 50% ACN, as evident from far-UV Circular Dichroism (CD) and Attenuated Total Reflection Fourier transformed infra red spectroscopy (ATR-FTIR). Tryptophan and 8-Anilino-1-Naphthalene-Sulphonic acid (ANS) fluorescence indicate altered tryptophan environment and high ANS binding suggesting a compact “molten globule”-like conformation with enhanced exposure of hydrophobic surface area. However, in presence of NaCl no intermediate state was observed. Detection of aggregates in HSA and OVA was possible at 90% ACN. Aggregates possess extensive β-sheet structure as revealed by far-UV CD and ATR-FTIR. These aggregates exhibit increase Thioflavin T (Th T) fluorescence with a red shift of Congo red (CR) absorption spectrum. X-ray diffraction (XRD) and Scanning Electron Microscopy (SEM) analysis confirmed the presence of fibrillar aggregates. Single cell gel electrophoresis (SCGE) assay of these fibrillar aggregates showed the DNA damage resulting in cell necrosis confirming their genotoxic nature. Some proteins not related to any human disease form fibrils in vitro. In the present study ACN gives access to a model system to study the process of aggregation. PMID:23342075

Influence of gold nanoparticles on platelets functional activity in vitro

NASA Astrophysics Data System (ADS)

Akchurin, Garif G.; Akchurin, George G.; Ivanov, Alexey N.; Kirichuk, Vyacheslav F.; Terentyuk, George S.; Khlebtsov, Boris N.; Khlebtsov, Nikolay G.

2008-02-01

Now in the leading biomedical centers of the world approved new technology of laser photothermal destruction of cancer cells using plasmon gold nanoparticles. Investigations of influence of gold nanoparticles on white rat platelets aggregative activity in vitro have been made. Platelet aggregation was investigated in platelet rich plasma (PRP) with help of laser analyzer 230 LA <>, Russia). Aggregation inductor was ADP solution in terminal concentration 2.5 micromole (<>, Russia). Gold nanoshells soluted in salt solution were used for experiments. Samples of PRP were incubated with 50 or 100 μl gold nanoshells solution in 5 minute, after that we made definition ADP induced platelet aggregation. We found out increase platelet function activity after incubation with nanoparticles solution which shown in maximum ADP-induced aggregation degree increase. Increase platelet function activity during intravenous nanoshells injection can be cause of thrombosis on patients. That's why before clinical application of cancer cell destruction based on laser photothermal used with plasmon gold nanoparticles careful investigations of thrombosis process and detail analyze of physiological blood parameters are very necessary.

Platelet activation and aggregation by the opportunistic pathogen Cutibacterium (Propionibacterium) acnes

PubMed Central

Petersson, Frida; Kilsgård, Ola; Shannon, Oonagh

2018-01-01

Cutibacterium (Propionibacterium) acnes, considered a part of the skin microbiota, is one of the most commonly isolated anaerobic bacteria from medical implants in contact with plasma. However, the precise interaction of C. acnes with blood cells and plasma proteins has not been fully elucidated. Herein, we have investigated the molecular interaction of C. acnes with platelets and plasma proteins. We report that the ability of C. acnes to aggregate platelets is dependent on phylotype, with a significantly lower ability amongst type IB isolates, and the interaction of specific donor-dependent plasma proteins (or concentrations thereof) with C. acnes. Pretreatment of C. acnes with plasma reduces the lag time before aggregation demonstrating that pre-deposition of plasma proteins on C. acnes is an important step in platelet aggregation. Using mass spectrometry we identified several plasma proteins deposited on C. acnes, including IgG, fibrinogen and complement factors. Inhibition of IgG, fibrinogen or complement decreased C. acnes-mediated platelet aggregation, demonstrating the importance of these plasma proteins for aggregation. The interaction of C. acnes and platelets was visualized using fluorescence microscopy, verifying the presence of IgG and fibrinogen as components of the aggregates, and co-localization of C. acnes and platelets in the aggregates. Here, we have demonstrated the ability of C. acnes to activate and aggregate platelets in a bacterium and donor-specific fashion, as well as added mechanistic insights into this interaction. PMID:29385206

Platelet activation and aggregation by the opportunistic pathogen Cutibacterium (Propionibacterium) acnes.

PubMed

Petersson, Frida; Kilsgård, Ola; Shannon, Oonagh; Lood, Rolf

2018-01-01

Cutibacterium (Propionibacterium) acnes, considered a part of the skin microbiota, is one of the most commonly isolated anaerobic bacteria from medical implants in contact with plasma. However, the precise interaction of C. acnes with blood cells and plasma proteins has not been fully elucidated. Herein, we have investigated the molecular interaction of C. acnes with platelets and plasma proteins. We report that the ability of C. acnes to aggregate platelets is dependent on phylotype, with a significantly lower ability amongst type IB isolates, and the interaction of specific donor-dependent plasma proteins (or concentrations thereof) with C. acnes. Pretreatment of C. acnes with plasma reduces the lag time before aggregation demonstrating that pre-deposition of plasma proteins on C. acnes is an important step in platelet aggregation. Using mass spectrometry we identified several plasma proteins deposited on C. acnes, including IgG, fibrinogen and complement factors. Inhibition of IgG, fibrinogen or complement decreased C. acnes-mediated platelet aggregation, demonstrating the importance of these plasma proteins for aggregation. The interaction of C. acnes and platelets was visualized using fluorescence microscopy, verifying the presence of IgG and fibrinogen as components of the aggregates, and co-localization of C. acnes and platelets in the aggregates. Here, we have demonstrated the ability of C. acnes to activate and aggregate platelets in a bacterium and donor-specific fashion, as well as added mechanistic insights into this interaction.

Construction of bioengineered hepatic tissue derived from human umbilical cord mesenchymal stem cells via aggregation culture in porcine decellularized liver scaffolds.

PubMed

Li, Yi; Wu, Qiong; Wang, Yujia; Li, Li; Chen, Fei; Shi, Yujun; Bao, Ji; Bu, Hong

2017-01-01

An individualized, tissue-engineered liver suitable for transplanting into a patient with liver disease would be of great benefit to the patient and the healthcare system. The tissue-engineered liver would possess the functions of the original healthy organ. Two fields of study, (i) using decellularized tissue as cell scaffolding, and (ii) stem cell differentiation into functional cells, are coming together to make this concept feasible. The decellularized liver scaffolds (DLS) can interact with cells to promote cell differentiation and signal transduction and three-dimensional (3D) stem cell aggregations can maintain the phenotypes and improve functions of stem cells after differentiation by undergoing cell-cell contact. Although the effects of DLS and stem cell aggregation culture have been intensively studied, few observations about the interaction between the two have been achieved. We established a method that combines the use of decellularized liver scaffolds and aggregation culture of MSCs (3D-DLS) and explored the effects of the two on hepatic differentiation of human umbilical cord mesenchymal stem cells (hUC-MSCs) in bioengineered hepatic tissue. A higher percentage of albumin-producing cells, higher levels of liver-specific transcripts, higher urea cycle-related transcripts, and lower levels of stem cell-specific transcripts were observed in the 3D-DLS group when compared to that of hUC-MSCs in monolayer culture (2D), aggregation culture (3D), monolayer on DLS culture (2D-DLS). The gene arrays also indicated that 3D-DLS induced the differentiation from the hUC-MSC phenotype to the PHH phenotype. Liver-specific proteins albumin, CK-18, and glycogen storage were highly positive in the 3D-DLS group. Albumin secretion and ammonia conversion to urea were more effective with a higher cell survival rate in the 3D-DLS group for 14 days. This DLS and aggregation combination culture system provides a novel method to improve hepatic differentiation, maintain phenotype of hepatocyte-like cells and sustain survival for 14 days in vitro. This is a promising strategy to use to construct bioengineered hepatic tissue. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Hsp72 (HSPA1A) Prevents Human Islet Amyloid Polypeptide Aggregation and Toxicity: A New Approach for Type 2 Diabetes Treatment

PubMed Central

Rosas, Paola C.; Nagaraja, Ganachari M.; Kaur, Punit; Panossian, Alexander; Wickman, Georg; Garcia, L. Rene; Al-Khamis, Fahd A.; Asea, Alexzander A. A.

2016-01-01

Type 2 diabetes is a growing public health concern and accounts for approximately 90% of all the cases of diabetes. Besides insulin resistance, type 2 diabetes is characterized by a deficit in β-cell mass as a result of misfolded human islet amyloid polypeptide (h-IAPP) which forms toxic aggregates that destroy pancreatic β-cells. Heat shock proteins (HSP) play an important role in combating the unwanted self-association of unfolded proteins. We hypothesized that Hsp72 (HSPA1A) prevents h-IAPP aggregation and toxicity. In this study, we demonstrated that thermal stress significantly up-regulates the intracellular expression of Hsp72, and prevents h-IAPP toxicity against pancreatic β-cells. Moreover, Hsp72 (HSPA1A) overexpression in pancreatic β-cells ameliorates h-IAPP toxicity. To test the hypothesis that Hsp72 (HSPA1A) prevents aggregation and fibril formation, we established a novel C. elegans model that expresses the highly amyloidogenic human pro-IAPP (h-proIAPP) that is implicated in amyloid formation and β-cell toxicity. We demonstrated that h-proIAPP expression in body-wall muscles, pharynx and neurons adversely affects C. elegans development. In addition, we demonstrated that h-proIAPP forms insoluble aggregates and that the co-expression of h-Hsp72 in our h-proIAPP C. elegans model, increases h-proIAPP solubility. Furthermore, treatment of transgenic h-proIAPP C. elegans with ADAPT-232, known to induce the expression and release of Hsp72 (HSPA1A), significantly improved the growth retardation phenotype of transgenic worms. Taken together, this study identifies Hsp72 (HSPA1A) as a potential treatment to prevent β-cell mass decline in type 2 diabetic patients and establishes for the first time a novel in vivo model that can be used to select compounds that attenuate h-proIAPP aggregation and toxicity. PMID:26960140

Hsp72 (HSPA1A) Prevents Human Islet Amyloid Polypeptide Aggregation and Toxicity: A New Approach for Type 2 Diabetes Treatment.

PubMed

Rosas, Paola C; Nagaraja, Ganachari M; Kaur, Punit; Panossian, Alexander; Wickman, Georg; Garcia, L Rene; Al-Khamis, Fahd A; Asea, Alexzander A A

2016-01-01

Type 2 diabetes is a growing public health concern and accounts for approximately 90% of all the cases of diabetes. Besides insulin resistance, type 2 diabetes is characterized by a deficit in β-cell mass as a result of misfolded human islet amyloid polypeptide (h-IAPP) which forms toxic aggregates that destroy pancreatic β-cells. Heat shock proteins (HSP) play an important role in combating the unwanted self-association of unfolded proteins. We hypothesized that Hsp72 (HSPA1A) prevents h-IAPP aggregation and toxicity. In this study, we demonstrated that thermal stress significantly up-regulates the intracellular expression of Hsp72, and prevents h-IAPP toxicity against pancreatic β-cells. Moreover, Hsp72 (HSPA1A) overexpression in pancreatic β-cells ameliorates h-IAPP toxicity. To test the hypothesis that Hsp72 (HSPA1A) prevents aggregation and fibril formation, we established a novel C. elegans model that expresses the highly amyloidogenic human pro-IAPP (h-proIAPP) that is implicated in amyloid formation and β-cell toxicity. We demonstrated that h-proIAPP expression in body-wall muscles, pharynx and neurons adversely affects C. elegans development. In addition, we demonstrated that h-proIAPP forms insoluble aggregates and that the co-expression of h-Hsp72 in our h-proIAPP C. elegans model, increases h-proIAPP solubility. Furthermore, treatment of transgenic h-proIAPP C. elegans with ADAPT-232, known to induce the expression and release of Hsp72 (HSPA1A), significantly improved the growth retardation phenotype of transgenic worms. Taken together, this study identifies Hsp72 (HSPA1A) as a potential treatment to prevent β-cell mass decline in type 2 diabetic patients and establishes for the first time a novel in vivo model that can be used to select compounds that attenuate h-proIAPP aggregation and toxicity.

Effects of Aggregation on Blood Sedimentation and Conductivity

PubMed Central

Zhbanov, Alexander; Yang, Sung

2015-01-01

The erythrocyte sedimentation rate (ESR) test has been used for over a century. The Westergren method is routinely used in a variety of clinics. However, the mechanism of erythrocyte sedimentation remains unclear, and the 60 min required for the test seems excessive. We investigated the effects of cell aggregation during blood sedimentation and electrical conductivity at different hematocrits. A sample of blood was drop cast into a small chamber with two planar electrodes placed on the bottom. The measured blood conductivity increased slightly during the first minute and decreased thereafter. We explored various methods of enhancing or retarding the erythrocyte aggregation. Using experimental measurements and theoretical calculations, we show that the initial increase in blood conductivity was indeed caused by aggregation, while the subsequent decrease in conductivity resulted from the deposition of erythrocytes. We present a method for calculating blood conductivity based on effective medium theory. Erythrocytes are modeled as conducting spheroids surrounded by a thin insulating membrane. A digital camera was used to investigate the erythrocyte sedimentation behavior and the distribution of the cell volume fraction in a capillary tube. Experimental observations and theoretical estimations of the settling velocity are provided. We experimentally demonstrate that the disaggregated cells settle much slower than the aggregated cells. We show that our method of measuring the electrical conductivity credibly reflected the ESR. The method was very sensitive to the initial stage of aggregation and sedimentation, while the sedimentation curve for the Westergren ESR test has a very mild slope in the initial time. We tested our method for rapid estimation of the Westergren ESR. We show a correlation between our method of measuring changes in blood conductivity and standard Westergren ESR method. In the future, our method could be examined as a potential means of accelerating ESR tests in clinical practice. PMID:26047511

RasC is required for optimal activation of adenylyl cyclase and Akt/PKB during aggregation

PubMed Central

Lim, Chinten James; Spiegelman, George B.; Weeks, Gerald

2001-01-01

Disruption of Dictyostelium rasC, encoding a Ras subfamily protein, generated cells incapable of aggregation. While rasC expression is enriched in a cell type-specific manner during post-aggregative development, the defect in rasC– cells is restricted to aggregation and fully corrected by application of exogenous cAMP pulses. cAMP is not produced in rasC– cells stimulated by 2′-deoxy-cAMP, but is produced in response to GTPγS in cell lysates, indicating that G-protein-coupled cAMP receptor activation of adenylyl cyclase is regulated by RasC. However, cAMP-induced ERK2 phosphorylation is unaffected in rasC– cells, indicating that RasC is not an upstream activator of the mitogen-activated protein kinase required for cAMP relay. rasC– cells also exhibit reduced chemotaxis to cAMP during early development and delayed response to periodic cAMP stimuli produced by wild-type cells in chimeric mixtures. Furthermore, cAMP-induced Akt/PKB phosphorylation through a phosphatidylinositide 3-kinase (PI3K)-dependent pathway is dramatically reduced in rasC– cells, suggesting that G-protein-coupled serpentine receptor activation of PI3K is regulated by RasC. Cells lacking the RasGEF, AleA, exhibit similar defects as rasC– cells, suggesting that AleA may activate RasC. PMID:11500376

RasC is required for optimal activation of adenylyl cyclase and Akt/PKB during aggregation.

PubMed

Lim, C J; Spiegelman, G B; Weeks, G

2001-08-15

Disruption of Dictyostelium rasC, encoding a Ras subfamily protein, generated cells incapable of aggregation. While rasC expression is enriched in a cell type-specific manner during post-aggregative development, the defect in rasC(-) cells is restricted to aggregation and fully corrected by application of exogenous cAMP pulses. cAMP is not produced in rasC(-) cells stimulated by 2'-deoxy-cAMP, but is produced in response to GTPgammaS in cell lysates, indicating that G-protein-coupled cAMP receptor activation of adenylyl cyclase is regulated by RasC. However, cAMP-induced ERK2 phosphorylation is unaffected in rasC(-) cells, indicating that RasC is not an upstream activator of the mitogen-activated protein kinase required for cAMP relay. rasC(-) cells also exhibit reduced chemotaxis to cAMP during early development and delayed response to periodic cAMP stimuli produced by wild-type cells in chimeric mixtures. Furthermore, cAMP-induced Akt/PKB phosphorylation through a phosphatidylinositide 3-kinase (PI3K)-dependent pathway is dramatically reduced in rasC(-) cells, suggesting that G-protein-coupled serpentine receptor activation of PI3K is regulated by RasC. Cells lacking the RasGEF, AleA, exhibit similar defects as rasC(-) cells, suggesting that AleA may activate RasC.

Cryopreservation of embryonic stem cell-derived multicellular neural aggregates labeled with micron-sized particles of iron oxide for magnetic resonance imaging.

PubMed

Yan, Yuanwei; Sart, Sébastien; Calixto Bejarano, Fabian; Muroski, Megan E; Strouse, Geoffrey F; Grant, Samuel C; Li, Yan

2015-01-01

Magnetic resonance imaging (MRI) provides an effective approach to track labeled pluripotent stem cell (PSC)-derived neural progenitor cells (NPCs) for neurological disorder treatments after cell labeling with a contrast agent, such as an iron oxide derivative. Cryopreservation of pre-labeled neural cells, especially in three-dimensional (3D) structure, can provide a uniform cell population and preserve the stem cell niche for the subsequent applications. In this study, the effects of cryopreservation on PSC-derived multicellular NPC aggregates labeled with micron-sized particles of iron oxide (MPIO) were investigated. These NPC aggregates were labeled prior to cryopreservation because labeling thawed cells can be limited by inefficient intracellular uptake, variations in labeling efficiency, and increased culture time before use, minimizing their translation to clinical settings. The results indicated that intracellular MPIO incorporation was retained after cryopreservation (70-80% labeling efficiency), and MPIO labeling had little adverse effects on cell recovery, proliferation, cytotoxicity and neural lineage commitment post-cryopreservation. MRI analysis showed comparable detectability for the MPIO-labeled cells before and after cryopreservation indicated by T2 and T2* relaxation rates. Cryopreserving MPIO-labeled 3D multicellular NPC aggregates can be applied in in vivo cell tracking studies and lead to more rapid translation from preservation to clinical implementation. © 2015 American Institute of Chemical Engineers.

Large SOD1 aggregates, unlike trimeric SOD1, do not impact cell viability in a model of amyotrophic lateral sclerosis.

PubMed

Zhu, Cheng; Beck, Matthew V; Griffith, Jack D; Deshmukh, Mohanish; Dokholyan, Nikolay V

2018-05-01

Aberrant accumulation of misfolded Cu, Zn superoxide dismutase (SOD1) is a hallmark of SOD1-associated amyotrophic lateral sclerosis (ALS), an invariably fatal neurodegenerative disease. While recent discovery of nonnative trimeric SOD1-associated neurotoxicity has suggested a potential pathway for motor neuron impairment, it is yet unknown whether large, insoluble aggregates are cytotoxic. Here we designed SOD1 mutations that specifically stabilize either the fibrillar form or the trimeric state of SOD1. The designed mutants display elevated populations of fibrils or trimers correspondingly, as demonstrated by gel filtration chromatography and electron microscopy. The trimer-stabilizing mutant, G147P, promoted cell death, even more potently in comparison with the aggressive ALS-associated mutants A4V and G93A. In contrast, the fibril-stabilizing mutants, N53I and D101I, positively impacted the survival of motor neuron-like cells. Hence, we conclude the SOD1 oligomer and not the mature form of aggregated fibril is critical for the neurotoxic effects in the model of ALS. The formation of large aggregates is in competition with trimer formation, suggesting that aggregation may be a protective mechanism against formation of toxic oligomeric intermediates.

Multilayer Dye Aggregation at Dye/TiO2 Interface via π…π Stacking and Hydrogen Bond and Its Impact on Solar Cell Performance: A DFT Analysis.

PubMed

Zhang, Lei; Liu, Xiaogang; Rao, Weifeng; Li, Jingfa

2016-10-21

Multilayer dye aggregation at the dye/TiO 2 interface of dye-sensitized solar cells is probed via first principles calculations, using p-methyl red azo dye as an example. Our calculations suggest that the multilayer dye aggregates at the TiO 2 surface can be stabilized by π…π stacking and hydrogen bond interactions. Compared with previous two-dimensional monolayer dye/TiO 2 model, the multilayer dye aggregation model proposed in this study constructs a three-dimensional multilayer dye/TiO 2 interfacial structure, and provides a better agreement between experimental and computational results in dye coverage and dye adsorption energy. In particular, a dimer forms by π…π stacking interactions between two neighboring azo molecules, while one of them chemisorbs on the TiO 2 surface; a trimer may form by introducing one additional azo molecule on the dimer through a hydrogen bond between two carboxylic acid groups. Different forms of multilayer dye aggregates, either stabilized by π…π stacking or hydrogen bond, exhibit varied optical absorption spectra and electronic properties. Such variations could have a critical impact on the performance of dye sensitized solar cells.

The inhibition of IGF-1 signaling promotes proteostasis by enhancing protein aggregation and deposition.

PubMed

Moll, Lorna; Ben-Gedalya, Tziona; Reuveni, Hadas; Cohen, Ehud

2016-04-01

The discovery that the alteration of aging by reducing the activity of the insulin/IGF-1 signaling (IIS) cascade protects nematodes and mice from neurodegeneration-linked, toxic protein aggregation (proteotoxicity) raises the prospect that IIS inhibitors bear therapeutic potential to counter neurodegenerative diseases. Recently, we reported that NT219, a highly efficient IGF-1 signaling inhibitor, protects model worms from the aggregation of amyloid β peptide and polyglutamine peptides that are linked to the manifestation of Alzheimer's and Huntington's diseases, respectively. Here, we employed cultured cell systems to investigate whether NT219 promotes protein homeostasis (proteostasis) in mammalian cells and to explore its underlying mechanisms. We found that NT219 enhances the aggregation of misfolded prion protein and promotes its deposition in quality control compartments known as "aggresomes." NT219 also elevates the levels of certain molecular chaperones but, surprisingly, reduces proteasome activity and impairs autophagy. Our findings show that IGF-1 signaling inhibitors in general and NT219 in particular can promote proteostasis in mammalian cells by hyperaggregating hazardous proteins, thereby bearing the potential to postpone the onset and slow the progression of neurodegenerative illnesses in the elderly.-Moll, L., Ben-Gedalya, T., Reuveni, H., Cohen, E. The inhibition of IGF-1 signaling promotes proteostasis by enhancing protein aggregation and deposition. © FASEB.

Multilayer Dye Aggregation at Dye/TiO2 Interface via π…π Stacking and Hydrogen Bond and Its Impact on Solar Cell Performance: A DFT Analysis

PubMed Central

Zhang, Lei; Liu, Xiaogang; Rao, Weifeng; Li, Jingfa

2016-01-01

Multilayer dye aggregation at the dye/TiO2 interface of dye-sensitized solar cells is probed via first principles calculations, using p-methyl red azo dye as an example. Our calculations suggest that the multilayer dye aggregates at the TiO2 surface can be stabilized by π…π stacking and hydrogen bond interactions. Compared with previous two-dimensional monolayer dye/TiO2 model, the multilayer dye aggregation model proposed in this study constructs a three-dimensional multilayer dye/TiO2 interfacial structure, and provides a better agreement between experimental and computational results in dye coverage and dye adsorption energy. In particular, a dimer forms by π…π stacking interactions between two neighboring azo molecules, while one of them chemisorbs on the TiO2 surface; a trimer may form by introducing one additional azo molecule on the dimer through a hydrogen bond between two carboxylic acid groups. Different forms of multilayer dye aggregates, either stabilized by π…π stacking or hydrogen bond, exhibit varied optical absorption spectra and electronic properties. Such variations could have a critical impact on the performance of dye sensitized solar cells. PMID:27767196

In vitro generation of three-dimensional substrate-adherent embryonic stem cell-derived neural aggregates for application in animal models of neurological disorders.

PubMed

Hargus, Gunnar; Cui, Yi-Fang; Dihné, Marcel; Bernreuther, Christian; Schachner, Melitta

2012-05-01

In vitro-differentiated embryonic stem (ES) cells comprise a useful source for cell replacement therapy, but the efficiency and safety of a translational approach are highly dependent on optimized protocols for directed differentiation of ES cells into the desired cell types in vitro. Furthermore, the transplantation of three-dimensional ES cell-derived structures instead of a single-cell suspension may improve graft survival and function by providing a beneficial microenvironment for implanted cells. To this end, we have developed a new method to efficiently differentiate mouse ES cells into neural aggregates that consist predominantly (>90%) of postmitotic neurons, neural progenitor cells, and radial glia-like cells. When transplanted into the excitotoxically lesioned striatum of adult mice, these substrate-adherent embryonic stem cell-derived neural aggregates (SENAs) showed significant advantages over transplanted single-cell suspensions of ES cell-derived neural cells, including improved survival of GABAergic neurons, increased cell migration, and significantly decreased risk of teratoma formation. Furthermore, SENAs mediated functional improvement after transplantation into animal models of Parkinson's disease and spinal cord injury. This unit describes in detail how SENAs are efficiently derived from mouse ES cells in vitro and how SENAs are isolated for transplantation. Furthermore, methods are presented for successful implantation of SENAs into animal models of Huntington's disease, Parkinson's disease, and spinal cord injury to study the effects of stem cell-derived neural aggregates in a disease context in vivo.

The Heteroaryldihydropyrimidine Bay 38-7690 Induces Hepatitis B Virus Core Protein Aggregates Associated with Promyelocytic Leukemia Nuclear Bodies in Infected Cells.

PubMed

Huber, Andrew D; Wolf, Jennifer J; Liu, Dandan; Gres, Anna T; Tang, Jing; Boschert, Kelsey N; Puray-Chavez, Maritza N; Pineda, Dallas L; Laughlin, Thomas G; Coonrod, Emily M; Yang, Qiongying; Ji, Juan; Kirby, Karen A; Wang, Zhengqiang; Sarafianos, Stefan G

2018-04-25

Heteroaryldihydropyrimidines (HAPs) are compounds that inhibit hepatitis B virus (HBV) replication by modulating viral capsid assembly. While their biophysical effects on capsid assembly in vitro have been previously studied, the effect of HAP treatment on capsid protein (Cp) in individual HBV-infected cells remains unknown. We report here that the HAP Bay 38-7690 promotes aggregation of recombinant Cp in vitro and causes a time- and dose-dependent decrease of Cp in infected cells, consistent with previously studied HAPs. Interestingly, immunofluorescence analysis showed Cp aggregating in nuclear foci of Bay 38-7690-treated infected cells in a time- and dose-dependent manner. We found these foci to be associated with promyelocytic leukemia (PML) nuclear bodies (NBs), which are structures that affect many cellular functions, including DNA damage response, transcription, apoptosis, and antiviral responses. Cp aggregation is not an artifact of the cell system used, as it is observed in HBV-expressing HepAD38 cells, in HepG2 cells transfected with an HBV-expressing plasmid, and in HepG2-NTCP cells infected with HBV. Use of a Cp overexpression vector without HBV sequences shows that aggregation is independent of viral replication, and use of an HBV-expressing plasmid harboring a HAP resistance mutation in Cp abrogated the aggregation, demonstrating that the effect is due to direct compound-Cp interactions. These studies provide novel insight into the effects of HAP-based treatment at a single-cell level. IMPORTANCE Despite the availability of effective vaccines and treatments, HBV remains a significant global health concern, with more than 240 million individuals chronically infected. Current treatments are highly effective at controlling viral replication and disease progression but rarely cure infections. Therefore, much emphasis is being placed on finding therapeutics with new drug targets, such as viral gene expression, covalently closed circular DNA formation and stability, capsid formation, and host immune modulators, with the ultimate goal of an HBV cure. Understanding the mechanisms by which novel antiviral agents act will be imperative for the development of curative HBV therapies. Copyright © 2018 Huber et al.

FYCO1 mediates clearance of α-synuclein aggregates through a Rab7-dependent mechanism.

PubMed

Saridaki, Theodora; Nippold, Markus; Dinter, Elisabeth; Roos, Andreas; Diederichs, Leonie; Fensky, Luisa; Schulz, Jörg B; Falkenburger, Björn H

2018-05-10

Parkinson disease can be caused by mutations in the α-synuclein gene and is characterized by aggregates of α-synuclein protein. We have previously shown that overexpression of the small GTPase Rab7 can induce clearance of α-synuclein aggregates. In this study, we investigate which Rab7 effectors mediate this effect. To model Parkinson disease we expressed the pathogenic A53T mutant of α-synuclein in HEK293T cells and Drosophila melanogaster. We tested the Rab7 effectors FYVE and coiled-coil domain-containing protein 1 (FYCO1) and Rab-interacting lysosomal protein (RILP). FYCO1-EGFP decorated vesicles containing α-synuclein. RILP-EGFP also decorated vesicular structures, but they did not contain α-synuclein. FYCO1 overexpression reduced the number of cells with α-synuclein aggregates, defined as visible particles of EGFP-tagged α-synuclein, whereas RILP did not. FYCO1 but not RILP reduced the amount of α-synuclein protein as assayed by western blot, increased the disappearance of α-synuclein aggregates in time-lapse microscopy, and decreased α-synuclein-induced toxicity assayed by the Trypan blue assay. siRNA-mediated knockdown of FYCO1 but not RILP reduced Rab7 induced aggregate clearance. Collectively, these findings indicate that FYCO1 and not RILP mediates Rab7 induced aggregate clearance. The effect of FYCO1 on aggregate clearance was blocked by the dominant negative Rab7 indicating that FYCO1 requires active Rab7 to function. Electron microscopic analysis and insertion of lysosomal membranes into the plasma membrane indicate that FYCO1 could lead to secretion of α-synuclein aggregates. Extracellular α-synuclein as assayed by ELISA was, however, not increased with FYCO1. Coexpression of FYCO1 in the fly model decreased α-synuclein aggregates as shown by the filter trap assay and rescued the locomotor deficit resulting from neuronal A53T-α-synuclein expression. This latter finding confirms that a pathway involving Rab7 and FYCO1 stimulates degradation of α-synuclein and could be beneficial in patients with Parkinson disease. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Investigating the feasibility of scale up and automation of human induced pluripotent stem cells cultured in aggregates in feeder free conditions☆

PubMed Central

Soares, Filipa A.C.; Chandra, Amit; Thomas, Robert J.; Pedersen, Roger A.; Vallier, Ludovic; Williams, David J.

2014-01-01

The transfer of a laboratory process into a manufacturing facility is one of the most critical steps required for the large scale production of cell-based therapy products. This study describes the first published protocol for scalable automated expansion of human induced pluripotent stem cell lines growing in aggregates in feeder-free and chemically defined medium. Cells were successfully transferred between different sites representative of research and manufacturing settings; and passaged manually and using the CompacT SelecT automation platform. Modified protocols were developed for the automated system and the management of cells aggregates (clumps) was identified as the critical step. Cellular morphology, pluripotency gene expression and differentiation into the three germ layers have been used compare the outcomes of manual and automated processes. PMID:24440272

Generation of chimeric minipigs by aggregating 4- to 8-cell-stage blastomeres from somatic cell nuclear transfer with the tracing of enhanced green fluorescent protein.

PubMed

Ji, Huili; Long, Chuan; Feng, Chong; Shi, Ningning; Jiang, Yingdi; Zeng, Guomin; Li, Xirui; Wu, Jingjing; Lu, Lin; Lu, Shengsheng; Pan, Dengke

2017-05-01

Blastocyst complementation is an important technique for generating chimeric organs in organ-deficient pigs, which holds great promise for solving the problem of a shortage of organs for human transplantation procedures. Porcine chimeras have been generated using embryonic germ cells, embryonic stem cells, and induced pluripotent stem cells; however, there are no authentic pluripotent stem cells for pigs. In previous studies, blastomeres from 4- to 8-cell-stage parthenogenetic embryos were able to generate chimeric fetuses efficiently, but the resulting fetuses did not produce live-born young. Here, we used early-stage embryos from somatic cell nuclear transfer (SCNT) to generate chimeric piglets by the aggregation method. Then, the distribution of chimerism in various tissues and organs was observed through the expression of enhanced green fluorescent protein (EGFP). Initially, we determined whether 4- to 8- or 8- to 16-cell-stage embryos were more suitable to generate chimeric piglets. Chimeras were produced by aggregating two EGFP-tagged Wuzhishan minipig (WZSP) SCNT embryos and two Bama minipig (BMP) SCNT embryos. The chimeric piglets were identified by coat color and microsatellite and swine leukocyte antigen analyses. Moreover, the distribution of chimerism in various tissues and organs of the piglets was evaluated by EGFP expression. We found that more aggregated embryos were produced using 4- to 8-cell-stage embryos (157/657, 23.9%) than 8- to 16-cell-stage embryos (100/499, 20.0%). Thus, 4- to 8-cell-stage embryos were used for the generation of chimeras. The rate of blastocysts development after aggregating WZSP with BMP embryos was 50.6%. Transfer of 391 blastocysts developed from 4- to 8-cell-stage embryos to five recipients gave rise to 18 piglets, of which two (11.1%) were confirmed to be chimeric by their coat color and microsatellite examination of the skin. One of the chimeric piglets died at 35 days and was subsequently autopsied, whereas the other piglet was maintained for the following observations. The heart and kidneys of the dead piglet showed chimerism, whereas the spinal cord, stomach, pancreas, intestines, muscle, ovary, and brain had no chimerism. To our knowledge, this is the first report of porcine chimeras generated by aggregating 4- to 8-cell-stage blastomeres from SCNT. We detected chimerism only in the skin, heart, and kidneys. Collectively, these results indicate that aggregation using 4- to 8-cell-stage SCNT embryos offers a practical approach for producing chimeric minipigs. Furthermore, it also provides a potential platform for generating interspecific chimeras between pigs and non-human primates for xenotransplantation. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Generating Porcine Chimeras Using Inner Cell Mass Cells and Parthenogenetic Preimplantation Embryos

PubMed Central

Nakano, Kazuaki; Watanabe, Masahito; Matsunari, Hitomi; Matsuda, Taisuke; Honda, Kasumi; Maehara, Miki; Kanai, Takahiro; Hayashida, Gota; Kobayashi, Mirina; Kuramoto, Momoko; Arai, Yoshikazu; Umeyama, Kazuhiro; Fujishiro, Shuh-hei; Mizukami, Yoshihisa; Nagaya, Masaki; Hanazono, Yutaka; Nagashima, Hiroshi

2013-01-01

Background The development and validation of stem cell therapies using induced pluripotent stem (iPS) cells can be optimized through translational research using pigs as large animal models, because pigs have the closest characteristics to humans among non-primate animals. As the recent investigations have been heading for establishment of the human iPS cells with naïve type characteristics, it is an indispensable challenge to develop naïve type porcine iPS cells. The pluripotency of the porcine iPS cells can be evaluated using their abilities to form chimeras. Here, we describe a simple aggregation method using parthenogenetic host embryos that offers a reliable and effective means of determining the chimera formation ability of pluripotent porcine cells. Methodology/Significant Principal Findings In this study, we show that a high yield of chimeric blastocysts can be achieved by aggregating the inner cell mass (ICM) from porcine blastocysts with parthenogenetic porcine embryos. ICMs cultured with morulae or 4–8 cell-stage parthenogenetic embryos derived from in vitro-matured (IVM) oocytes can aggregate to form chimeric blastocysts that can develop into chimeric fetuses after transfer. The rate of production of chimeric blastocysts after aggregation with host morulae (20/24, 83.3%) was similar to that after the injection of ICMs into morulae (24/29, 82.8%). We also found that 4–8 cell-stage embryos could be used; chimeric blastocysts were produced with a similar efficiency (17/26, 65.4%). After transfer into recipients, these blastocysts yielded chimeric fetuses at frequencies of 36.0% and 13.6%, respectively. Conclusion/Significance Our findings indicate that the aggregation method using parthenogenetic morulae or 4–8 cell-stage embryos offers a highly reproducible approach for producing chimeric fetuses from porcine pluripotent cells. This method provides a practical and highly accurate system for evaluating pluripotency of undifferentiated cells, such as iPS cells, based on their ability to form chimeras. PMID:23626746

Dynamic imaging of interaction between protein 14-3-3 and Bid in living cells

NASA Astrophysics Data System (ADS)

Chen, Tongsheng; Xing, Da; Wang, Jinjun

2006-02-01

The 14-3-3 proteins are known to sequester certain pro-apoptotic members of this family. BH3- interacting domain death agonist (Bid) may contribute to tumor necrosis factor α(TNF-α)-induced neuronal death, although regulation by 14-3-3 has not been reported. In this study we examined whether 14-3-3 proteins interact with Bid/tBid during TNF-α-induced cell death. The TNF-αtriggered Bid cleavage and tBid translocated to mitochondria. Human lung adenocarcinoma cells were co-transfected with both CFP-Bid and 14-3-3-YFP plasmids, and the dynamical interaction between the Bid/tBid and 14-3-3 were performed on laser confocal fluorescence microscope in single living cell during TNF-α-induced cell apoptosis. The Bid distribute equally only in the cytoplasm of healthy cells, and the 14-3-3 protein distribute not only in the cytoplasm but also in the nucleus of healthy cells. Our data showed that the tBid aggregate, but the 14-3-3 protein does not aggregate as the tBid, and the 14-3-3 protein separate from the aggregated tBid, implying that the 14-3-3 proteins do not interact with the aggregated tBid after TNF-αtreatment.

Dynamics and mechanisms of asbestos-fiber aggregate growth in water

NASA Astrophysics Data System (ADS)

Wu, L.; Ortiz, C. P.; Jerolmack, D. J.

2015-12-01

Most colloidal particles including asbestos fibers form aggregates in water, when solution chemistry provides favorable conditions. To date, the growth of colloidal aggregates has been observed in many model systems under optical and scanning electron microscopy; however, all of these studies have used near-spherical particles. The highly elongated nature of asbestos fibers may cause anomalous aggregate growth and morphology, but this has never been examined. Although the exposure pathway of concern for asbestos is through the air, asbestos particles typically reside in soil that is at least partially saturated, and aggregates formed in the aqueous phase may influence the mobility of particles in the environment. Here we study solution-phase aggregation kinetics of asbestos fibers using a liquid-cell by in situ microscopy, over micron to centimeter length scales and from a tenth of a second to hours. We employ an elliptical particle tracking technique to determine particle trajectories and to quantify diffusivity. Experiments reveal that diffusing fibers join by cross linking, but that such linking is sometimes reversible. The resulting aggregates are very sparse and non-compact, with a fractal dimension that is lower than any previously reported value. Their morphology, growth rate and particle size distribution exhibit non-classical behavior that deviates significantly from observations of aggregates composed of near-spherical particles. We also perform experiments using synthetic colloidal particles, and compare these to asbestos in order to separate the controls of particle shape vs. material properties. This direct method for quantitatively observing aggregate growth is a first step toward predicting asbestos fiber aggregate size distributions in the environment. Moreover, many emerging environmental contaminants - such as carbon nanotubes - are elongated colloids, and our work suggests that theories for aggregate growth may need to be modified in order to model these particles.

Aspirin-Mediated Acetylation Protects Against Multiple Neurodegenerative Pathologies by Impeding Protein Aggregation.

PubMed

Ayyadevara, Srinivas; Balasubramaniam, Meenakshisundaram; Kakraba, Samuel; Alla, Ramani; Mehta, Jawahar L; Shmookler Reis, Robert J

2017-12-10

Many progressive neurological disorders, including Alzheimer's disease (AD), Huntington's disease, and Parkinson's disease (PD), are characterized by accumulation of insoluble protein aggregates. In prospective trials, the cyclooxygenase inhibitor aspirin (acetylsalicylic acid) reduced the risk of AD and PD, as well as cardiovascular events and many late-onset cancers. Considering the role played by protein hyperphosphorylation in aggregation and neurodegenerative diseases, and aspirin's known ability to donate acetyl groups, we asked whether aspirin might reduce both phosphorylation and aggregation by acetylating protein targets. Aspirin was substantially more effective than salicylate in reducing or delaying aggregation in human neuroblastoma cells grown in vitro, and in Caenorhabditis elegans models of human neurodegenerative diseases in vivo. Aspirin acetylates many proteins, while reducing phosphorylation, suggesting that acetylation may oppose phosphorylation. Surprisingly, acetylated proteins were largely excluded from compact aggregates. Molecular-dynamic simulations indicate that acetylation of amyloid peptide energetically disfavors its association into dimers and octamers, and oligomers that do form are less compact and stable than those comprising unacetylated peptides. Hyperphosphorylation predisposes certain proteins to aggregate (e.g., tau, α-synuclein, and transactive response DNA-binding protein 43 [TDP-43]), and it is a critical pathogenic marker in both cardiovascular and neurodegenerative diseases. We present novel evidence that acetylated proteins are underrepresented in protein aggregates, and that aggregation varies inversely with acetylation propensity after diverse genetic and pharmacologic interventions. These results are consistent with the hypothesis that aspirin inhibits protein aggregation and the ensuing toxicity of aggregates through its acetyl-donating activity. This mechanism may contribute to the neuro-protective, cardio-protective, and life-prolonging effects of aspirin. Antioxid. Redox Signal. 27, 1383-1396.

Distal Renal Tubules Are Deficient in Aggresome Formation and Autophagy upon Aldosterone Administration

PubMed Central

Cheema, Muhammad Umar; Damkier, Helle Hasager; Nielsen, Jakob; Poulsen, Ebbe Toftgaard; Enghild, Jan J.; Fenton, Robert A.; Praetorius, Jeppe

2014-01-01

Prolonged elevations of plasma aldosterone levels are associated with renal pathogenesis. We hypothesized that renal distress could be imposed by an augmented aldosterone-induced protein turnover challenging cellular protein degradation systems of the renal tubular cells. Cellular accumulation of specific protein aggregates in rat kidneys was assessed after 7 days of aldosterone administration. Aldosterone induced intracellular accumulation of 60 s ribosomal protein L22 in protein aggregates, specifically in the distal convoluted tubules. The mineralocorticoid receptor inhibitor spironolactone abolished aldosterone-induced accumulation of these aggregates. The aldosterone-induced protein aggregates also contained proteasome 20 s subunits. The partial de-ubiquitinase ataxin-3 was not localized to the distal renal tubule protein aggregates, and the aggregates only modestly colocalized with aggresome transfer proteins dynactin p62 and histone deacetylase 6. Intracellular protein aggregation in distal renal tubules did not lead to development of classical juxta-nuclear aggresomes or to autophagosome formation. Finally, aldosterone treatment induced foci in renal cortex of epithelial vimentin expression and a loss of E-cadherin expression, as signs of cellular stress. The cellular changes occurred within high, but physiological aldosterone concentrations. We conclude that aldosterone induces protein accumulation in distal renal tubules; these aggregates are not cleared by autophagy that may lead to early renal tubular damage. PMID:25000288

Non-pathogenic protein aggregates in skeletal muscle in MLF1 transgenic mice.

PubMed

Li, Zhi-Fang; Wu, Xiaohua; Jiang, Yun; Liu, Jianxiang; Wu, Chun; Inagaki, Masaki; Izawa, Ichiro; Mizisin, Andrew P; Engvall, Eva; Shelton, G Diane

2008-01-15

Protein aggregate formation in muscle is thought to be pathogenic and associated with clinical weakness. Over-expression of either wild type or a mutant form of myeloid leukemia factor 1 (MLF1) in transgenic mouse skeletal muscle and in cultured cells resulted in aggregate formation. Aggregates were detected in MLF1 transgenic mice at 6 weeks of age, and increased in size with age. However, histological examination of skeletal muscles of MLF1 transgenic mice revealed no pathological changes other than the aggregates, and RotaRod testing did not detect functional deficits. MLF1 has recently been identified as a protein that could neutralize the toxicity of intracellular protein aggregates in a Drosophila model of Huntington's disease (HD). We also demonstrate that MLF1 interacts with MRJ, a heat shock protein, which can independently neutralize the toxicity of intracellular protein aggregates in the Drosophila HD model. Our data suggest that over-expression of MLF1 has no significant impact on skeletal muscle function in mice; that progressive formation of protein aggregates in muscle are not necessarily pathogenic; and that MLF1 and MRJ may function together to ameliorate the toxic effects of polyglutamine or mutant proteins in myodegenerative diseases such as inclusion body myositis and oculopharyngeal muscular dystrophy, as well as neurodegenerative disease.

Fluorescence lifetime dynamics of enhanced green fluorescent protein in protein aggregates with expanded polyglutamine

NASA Astrophysics Data System (ADS)

Ghukasyan, Vladimir; Hsu, Chih-Chun; Liu, Chia-Rung; Kao, Fu-Jen; Cheng, Tzu-Hao

2010-01-01

Protein aggregation is one of the characteristic steps in a number of neurodegenerative diseases eventually leading to neuronal death and thorough study of aggregation is required for the development of effective therapy. We apply fluorescence lifetime imaging for the characterization of the fluorescence dynamics of the enhanced green fluorescent protein (eGFP) in fusion with the polyQ-expanded polyglutamine stretch. At the expansion of polyQ above 39 residues, it has an inherent propensity to form amyloid-like fibrils and aggregates, and is responsible for Huntington's disease. The results of the experiments show that expression of the eGFP in fusion with the 97Q protein leads to the decrease of the eGFP fluorescence lifetime by ~300 ps. This phenomenon does not appear in Hsp104-deficient cells, where the aggregation in polyQ is prevented. We demonstrate that the lifetime decrease observed is related to the aggregation per se and discuss the possible role of refractive index and homo-FRET in these dynamics.

Structure-activity relationship of cyanine tau aggregation inhibitors

PubMed Central

Chang, Edward; Congdon, Erin E.; Honson, Nicolette S.; Duff, Karen E.; Kuret, Jeff

2009-01-01

A structure-activity relationship for symmetrical cyanine inhibitors of human tau aggregation was elaborated using a filter trap assay. Antagonist activity depended on cyanine heterocycle, polymethine bridge length, and the nature of meso- and N-substituents. One potent member of the series, 3,3’-diethyl-9-methylthiacarbocyanine iodide (compound 11), retained submicromolar potency and had calculated physical properties consistent with blood-brain barrier and cell membrane penetration. Exposure of organotypic slices prepared from JNPL3 transgenic mice (which express human tau harboring the aggregation prone P301L tauopathy mutation) to compound 11 for one week revealed a biphasic dose response relationship. Low nanomolar concentrations decreased insoluble tau aggregates to half those observed in slices treated with vehicle alone. In contrast, high concentrations (≥300 nM) augmented tau aggregation and produced abnormalities in tissue tubulin levels. These data suggest that certain symmetrical carbocyanine dyes can modulate tau aggregation in the slice biological model at concentrations well below those associated with toxicity. PMID:19432420

An Interdomain Interaction of the Androgen Receptor Is Required for Its Aggregation and Toxicity in Spinal and Bulbar Muscular Atrophy*

PubMed Central

Orr, Christopher R.; Montie, Heather L.; Liu, Yuhong; Bolzoni, Elena; Jenkins, Shannon C.; Wilson, Elizabeth M.; Joseph, James D.; McDonnell, Donald P.; Merry, Diane E.

2010-01-01

Polyglutamine expansion within the androgen receptor (AR) causes spinal and bulbar muscular atrophy (SBMA) and is associated with misfolded and aggregated species of the mutant AR. We showed previously that nuclear localization of the mutant AR was necessary but not sufficient for SBMA. Here we show that an interdomain interaction of the AR that is central to its function within the nucleus is required for AR aggregation and toxicity. Ligands that prevent the interaction between the amino-terminal FXXLF motif and carboxyl-terminal AF-2 domain (N/C interaction) prevented toxicity and AR aggregation in an SBMA cell model and rescued primary SBMA motor neurons from 5α-dihydrotestosterone-induced toxicity. Moreover, genetic mutation of the FXXLF motif prevented AR aggregation and 5α-dihydrotestosterone toxicity. Finally, selective androgen receptor modulators, which prevent the N/C interaction, ameliorated AR aggregation and toxicity while maintaining AR function, highlighting a novel therapeutic strategy to prevent the SBMA phenotype while retaining AR transcriptional function. PMID:20826791

Control of binder viscosity and hygroscopicity on particle aggregation efficiency

NASA Astrophysics Data System (ADS)

Mueller, Sebastian B.; Kueppers, Ulrich; Ayris, Paul M.; Jacob, Michael; Delmelle, Pierre; Dingwell, Donald B.

2016-04-01

In the course of explosive volcanic eruptions, large amounts of ash are released into the atmosphere and may subsequently pose a threat to infrastructure, such as aviation industry. Ash plume forecasting is therefore a crucial tool for volcanic hazard mitigation but may be significantly affected by aggregation, altering the aerodynamic properties of particles. Models struggle with the implementation of aggregation since external conditions promoting aggregation have not been completely understood; in a previous study we have shown the rapid generation of ash aggregates through liquid bonding via the use of fluidization bed technology and further defined humidity and temperature ranges necessary to trigger aggregation. Salt (NaCl) was required for the recovery of stable aggregates, acting as a cementation agent and granting aggregate cohesion. A numerical model was used to explain the physics behind particle aggregation mechanisms and further predicted a dependency of aggregation efficiency on liquid binder viscosity. In this study we proof the effect of viscosity on particle aggregation. HCl and H2SO4 solutions were diluted to various concentrations resulting in viscosities between 1 and 2 mPas. Phonolitic and rhyolitic ash samples as well as soda-lime glass beads (serving as analogue material) were fluidized in the ProCell Lab® of Glatt Ingenieurtechnik GmbH and treated with the acids via a bottom-spray technique. Chemically driven interaction between acid liquids and surfaces of the three used materials led to crystal precipitation. Salt crystals (e.g. NaCl) have been confirmed through scanning electron microscopy (SEM) and leachate analysis. Both volcanic ash samples as well as the glass beads showed a clear dependency of aggregation efficiency on viscosity of the sprayed HCl solution. Spraying H2SO4 provoked a collapse of the fluidized bed and no aggregation has been observed. This is accounted by the high hygroscopicity of H2SO4. Dissolving CaCl2 (known to be a highly hygroscopic salt) in de-ionized water yielded comparable results without observable aggregation. In case of successful aggregation, concentration of salts has been found to be in the range of published values. We conclude that non-hygroscopic salt crystal precipitation from an aqueous liquid interacting with the glass phase in volcanic ash is a very efficient way to produce cohesive ash aggregates that can survive external forces acting during transport and sedimentation.

Isoform-specific PKA dynamics revealed by dye-triggered aggregation and DAKAP1alpha-mediated localization in living cells.

PubMed

Martin, Brent R; Deerinck, Thomas J; Ellisman, Mark H; Taylor, Susan S; Tsien, Roger Y

2007-09-01

The tetracysteine sequence YRECCPGCCMWR fused to the N terminus of green fluorescent protein (GFP) self-aggregates upon biarsenical labeling in living cells or in vitro. Such dye-triggered aggregates form temperature-dependent morphologies and are dispersed by photobleaching. Fusion of the biarsenical aggregating GFP to the regulatory (R) or catalytic (C) subunit of PKA traps intact holoenzyme in compact fluorescent puncta upon biarsenical labeling. Contrary to the classical model of PKA activation, elevated cAMP does not allow RIalpha and Calpha to diffuse far apart unless the pseudosubstrate inhibitor PKI or locally concentrated substrate is coexpressed. However, RIIalpha releases Calpha upon elevated cAMP alone, dependent on autophosphorylation of the RIIalpha inhibitory domain. DAKAP1alpha overexpression induced R and C outer mitochondrial colocalization and showed similar regulation. Overall, effective separation of type I PKA is substrate dependent, whereas type II PKA dissociation relies on autophosphorylation.

Biological role of bacterial inclusion bodies: a model for amyloid aggregation.

PubMed

García-Fruitós, Elena; Sabate, Raimon; de Groot, Natalia S; Villaverde, Antonio; Ventura, Salvador

2011-07-01

Inclusion bodies are insoluble protein aggregates usually found in recombinant bacteria when they are forced to produce heterologous protein species. These particles are formed by polypeptides that cross-interact through sterospecific contacts and that are steadily deposited in either the cell's cytoplasm or the periplasm. An important fraction of eukaryotic proteins form inclusion bodies in bacteria, which has posed major problems in the development of the biotechnology industry. Over the last decade, the fine dissection of the quality control system in bacteria and the recognition of the amyloid-like architecture of inclusion bodies have provided dramatic insights on the dynamic biology of these aggregates. We discuss here the relevant aspects, in the interface between cell physiology and structural biology, which make inclusion bodies unique models for the study of protein aggregation, amyloid formation and prion biology in a physiologically relevant background. © 2011 The Authors Journal compilation © 2011 FEBS.

Measurement of the temperature-dependent threshold shear-stress of red blood cell aggregation.

PubMed

Lim, Hyun-Jung; Nam, Jeong-Hun; Lee, Yong-Jin; Shin, Sehyun

2009-09-01

Red blood cell (RBC) aggregation is becoming an important hemorheological parameter, which typically exhibits temperature dependence. Quite recently, a critical shear-stress was proposed as a new dimensional index to represent the aggregative and disaggregative behaviors of RBCs. The present study investigated the effect of the temperature on the critical shear-stress that is required to keep RBC aggregates dispersed. The critical shear-stress was measured at various temperatures (4, 10, 20, 30, and 37 degrees C) through the use of a transient microfluidic aggregometry. The critical shear-stress significantly increased as the blood temperature lowered, which accorded with the increase in the low-shear blood viscosity with the lowering of the temperature. Furthermore, the critical shear-stress also showed good agreement with the threshold shear-stress, as measured in a rotational Couette flow. These findings assist in rheologically validating the critical shear-stress, as defined in the microfluidic aggregometry.

Experimental volcanic ash aggregation: Internal structuring of accretionary lapilli and the role of liquid bonding

NASA Astrophysics Data System (ADS)

Mueller, Sebastian B.; Kueppers, Ulrich; Ayris, Paul M.; Jacob, Michael; Dingwell, Donald B.

2016-01-01

Explosive volcanic eruptions can release vast quantities of pyroclastic material into Earth's atmosphere, including volcanic ash, particles with diameters less than two millimeters. Ash particles can cluster together to form aggregates, in some cases reaching up to several centimeters in size. Aggregation alters ash transport and settling behavior compared to un-aggregated particles, influencing ash distribution and deposit stratigraphy. Accretionary lapilli, the most commonly preserved type of aggregates within the geologic record, can exhibit complex internal stratigraphy. The processes involved in the formation and preservation of these aggregates remain poorly constrained quantitatively. In this study, we simulate the variable gas-particle flow conditions which may be encountered within eruption plumes and pyroclastic density currents via laboratory experiments using the ProCell Lab System® of Glatt Ingenieurtechnik GmbH. In this apparatus, solid particles are set into motion in a fluidized bed over a range of well-controlled boundary conditions (particle concentration, air flow rate, gas temperature, humidity, liquid composition). Experiments were conducted with soda-lime glass beads and natural volcanic ash particles under a range of experimental conditions. Both glass beads and volcanic ash exhibited the capacity for aggregation, but stable aggregates could only be produced when materials were coated with high but volcanically-relevant concentrations of NaCl. The growth and structure of aggregates was dependent on the initial granulometry, while the rate of aggregate formation increased exponentially with increasing relative humidity (12-45% RH), before overwetting promoted mud droplet formation. Notably, by use of a broad granulometry, we generated spherical, internally structured aggregates similar to some accretionary pellets found in volcanic deposits. Adaptation of a powder-technology model offers an explanation for the origin of natural accretionary pellets, suggesting them to be the result of a particular granulometry and fast-acting selective aggregation processes. For such aggregates to survive deposition and be preserved in the deposits of eruption plumes and pyroclastic density currents likely requires a significant pre-existing salt load on ash surfaces, and rapid aggregate drying prior to deposition or interaction with a more energetic environment. Our results carry clear benefits for future efforts to parameterize models of ash transport and deposition in the field.

ROCK and PRK-2 Mediate the Inhibitory Effect of Y-27632 on Polyglutamine Aggregation

PubMed Central

Shao, Jieya; Welch, William J.; Diamond, Marc I.

2009-01-01

Polyglutamine expansion in huntingtin (Htt) and the androgen receptor (AR) causes untreatable neurodegenerative diseases. Y-27632, a therapeutic lead, reduces Htt and AR aggregation in cultured cells, and Htt-induced neurodegeneration in Drosophila. Y-27632 inhibits both Rho-associated kinases ROCK and PRK-2, making its precise intracellular target uncertain. Over-expression of either kinase increases Htt and AR aggregation. Three ROCK inhibitors (Y-27632, H-1077, HA-1152), and a specific ROCK inhibitory peptide reduce polyglutamine protein aggregation, as does knockdown of ROCK or PRK-2 by RNAi. RNAi also indicates that each kinase is required for the inhibitory effects of Y-27632 to manifest fully. These two actin regulatory kinases are thus involved in polyglutamine aggregation, and their simultaneous inhibition may be an important therapeutic goal. PMID:18423405

A zeta potential value determines the aggregate's size of penta-substituted [60]fullerene derivatives in aqueous suspension whereas positive charge is required for toxicity against bacterial cells.

PubMed

Deryabin, Dmitry G; Efremova, Ludmila V; Vasilchenko, Alexey S; Saidakova, Evgeniya V; Sizova, Elena A; Troshin, Pavel A; Zhilenkov, Alexander V; Khakina, Ekaterina A; Khakina, Ekaterina E

2015-08-08

The cause-effect relationships between physicochemical properties of amphiphilic [60]fullerene derivatives and their toxicity against bacterial cells have not yet been clarified. In this study, we report how the differences in the chemical structure of organic addends in 10 originally synthesized penta-substituted [60]fullerene derivatives modulate their zeta potential and aggregate's size in salt-free and salt-added aqueous suspensions as well as how these physicochemical characteristics affect the bioenergetics of freshwater Escherichia coli and marine Photobacterium phosphoreum bacteria. Dynamic light scattering, laser Doppler micro-electrophoresis, agarose gel electrophoresis, atomic force microscopy, and bioluminescence inhibition assay were used to characterize the fullerene aggregation behavior in aqueous solution and their interaction with the bacterial cell surface, following zeta potential changes and toxic effects. Dynamic light scattering results indicated the formation of self-assembled [60]fullerene aggregates in aqueous suspensions. The measurement of the zeta potential of the particles revealed that they have different surface charges. The relationship between these physicochemical characteristics was presented as an exponential regression that correctly described the dependence of the aggregate's size of penta-substituted [60]fullerene derivatives in salt-free aqueous suspension from zeta potential value. The prevalence of DLVO-related effects was shown in salt-added aqueous suspension that decreased zeta potential values and affected the aggregation of [60]fullerene derivatives expressed differently for individual compounds. A bioluminescence inhibition assay demonstrated that the toxic effect of [60]fullerene derivatives against E. coli cells was strictly determined by their positive zeta potential charge value being weakened against P. phosphoreum cells in an aquatic system of high salinity. Atomic force microscopy data suggested that the activity of positively charged [60]fullerene derivatives against bacterial cells required their direct interaction. The following zeta potential inversion on the bacterial cells surface was observed as an early stage of toxicity mechanism that violates the membrane-associated energetic functions. The novel data about interrelations between physicochemical parameters and toxic properties of amphiphilic [60]fullerene derivatives make possible predicting their behavior in aquatic environment and their activity against bacterial cells.

Cell-free released components of Streptococcus sanguis inhibit human platelet aggregation.

PubMed Central

Herzberg, M C; Brintzenhofe, K L; Clawson, C C

1983-01-01

To study the role of surface components in the selective binding and aggregation of platelet-rich plasma (PRP) by strains of viridans streptococci, we treated the binding, aggregation strain Streptococcus sanguis I 2017-78 by sonication or trypsinization. Morphologically identifiable electron-dense fibrils were released from the cell wall, apparently from an inner electron-dense layer, under conditions that left cells intact. These controlled conditions were determined to cause submaximal loss in adhesion to platelet ghosts and PRP aggregation by treated, washed S. sanguis. Soluble components were recovered from the controlled sonic or L-(tosylamido 2-phenyl)ethyl chloromethyl ketone-trypsin treatments. Each showed dose-response inhibition of aggregation when preincubated with PRP before challenge with fresh, untreated S. sanguis. The time to onset of PRP aggregation was inhibited by 50% with 0.2 mg of TPCK-trypsin peptides or 1.0 mg of the sonicate per ml per 2 X 10(8) platelets. Components of both preparations were immunologically cross-reactive, but lipoteichoic acid was not a major antigen of either. By weight, the TPCK-trypsin peptides were virtually all protein; the sonicate residues identified were about 50% protein and 7% hexose. Each was a complex mixture of components as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. More than 8 TPCK-trypsin peptides and 16 sonicate components were so identified. In contrast, at least four or five components from either preparation were recognized as surface determinants by a rabbit antiserum to whole homologous microbes. Platelet-binding ligands of S. sanguis could be among these determinants. Images PMID:6618669

Detection of Mutant Huntingtin Aggregation Conformers and Modulation of SDS-Soluble Fibrillar Oligomers by Small Molecules

PubMed Central

Sontag, Emily Mitchell; Lotz, Gregor P.; Yang, Guocheng; Sontag, Christopher J.; Cummings, Brian J.; Glabe, Charles G.; Muchowski, Paul J.; Thompson, Leslie Michels

2012-01-01

The Huntington’s disease (HD) mutation leads to a complex process of Huntingtin (Htt) aggregation into multimeric species that eventually form visible inclusions in cytoplasm, nuclei and neuronal processes. One hypothesis is that smaller, soluble forms of amyloid proteins confer toxic effects and contribute to early cell dysfunction. However, analysis of mutant Htt aggregation intermediates to identify conformers that may represent toxic forms of the protein and represent potential drug targets remains difficult. We performed a detailed analysis of aggregation conformers in multiple in vitro, cell and ex vivo models of HD. Conformation-specific antibodies were used to identify and characterize aggregation species, allowing assessment of multiple conformers present during the aggregation process. Using a series of assays together with these antibodies, several forms could be identified. Fibrillar oligomers, defined as having a β-sheet rich conformation, are observed in vitro using recombinant protein and in protein extracts from cells in culture or mouse brain and shown to be globular, soluble and non-sedimentable structures. Compounds previously described to modulate visible inclusion body formation and reduce toxicity in HD models were also tested and consistently found to alter the formation of fibrillar oligomers. Interestingly, these compounds did not alter the rate of visible inclusion formation, indicating that fibrillar oligomers are not necessarily the rate limiting step of inclusion body formation. Taken together, we provide insights into the structure and formation of mutant Htt fibrillar oligomers that are modulated by small molecules with protective potential in HD models. PMID:24086178

Regulating Molecular Aggregations of Polymers via Ternary Copolymerization Strategy for Efficient Solar Cells.

PubMed

Wang, Qian; Wang, Yingying; Zheng, Wei; Shahid, Bilal; Qiu, Meng; Wang, Di; Zhu, Dangqiang; Yang, Renqiang

2017-09-20

For many high-performance photovoltaic materials in polymer solar cells (PSCs), the active layers usually need to be spin-coated at high temperature due to the strong intermolecular aggregation of donor polymers, which is unfavorable in device repeatability and large-scale PSC printing. In this work, we adopted a ternary copolymerization strategy to regulate polymer solubility and molecular aggregation. A series of D-A 1 -D-A 2 random polymers based on different acceptors, strong electron-withdrawing unit ester substituted thieno[3,4-b]thiophene (TT-E), and highly planar dithiazole linked TT-E (DTzTT) were constructed to realize the regulation of molecular aggregation and simplification of device fabrication. The results showed that as the relative proportion of TT-E segment in the backbone increased, the absorption evidently red-shifted with a gradually decreased aggregation in solution, eventually leading to the active layers that can be fabricated at low temperature. Furthermore, due to the excellent phase separation and low recombination, the optimized solar cells based on the terpolymer P1 containing 30% of TT-E segment exhibit high power conversion efficiency (PCE) of 9.09% with a significantly enhanced fill factor up to 72.86%. Encouragingly, the photovoltaic performance is insensitive to the fabrication temperature of the active layer, and it still could maintain high PCE of 8.82%, even at room temperature. This work not only develops the highly efficient photovoltaic materials for low temperature processed PSCs through ternary copolymerization strategy but also preliminarily constructs the relationship between aggregation and photovoltaic performance.

Fluorescent transgenic mice suitable for multi-color aggregation chimera studies.

PubMed

Ohtsuka, Masato; Miura, Hiromi; Gurumurthy, Channabasavaiah B; Kimura, Minoru; Inoko, Hidetoshi; Yoshimura, Shinichi; Sato, Masahiro

2012-11-01

We recently reported a novel method of mouse transgenesis called Pronuclear Injection-based Targeted Transgenisis (PITT) using which a series of fluorescent transgenic (Tg) mice lines were generated. These lines, unlike those generated using conventional random integration methods, express the transgenes faithfully and reproducibly generation after generation. Because of this superior nature, these lines are ideal for the generation of multi-colored aggregation chimeras that can be used to study cell-cell interactions and lineage analyses in living embryos/organs, where the transgenes can be detected and the clonal origin of a given cell population easily traced by its distinct fluorescence. In this study, to verify if Tg fluorescent mice generated through PITT were suitable for such applications, we sought to generate chimeric blastocysts and chimeric-Tg mice by aggregating two- or three-colored 8-cell embryos. Our analyses using these models led to the following observations. First, we noticed that cell mixing was infrequent during the stages of morula to early blastocyst. Second, chimeric fetuses obtained after aggregation of the two-colored 8-cell embryos exhibited uniform cell mixing. And third, in the organs of adult chimeric mice, the mode of cell distribution could be either clonal or polyclonal, as previously pointed out by others. Implications of our novel and improved Tg-chimeric mice approach for clonal cell lineage and developmental studies are discussed.

Reversal of a full-length mutant huntingtin neuronal cell phenotype by chemical inhibitors of polyglutamine-mediated aggregation

PubMed Central

Wang, Jin; Gines, Silvia; MacDonald, Marcy E; Gusella, James F

2005-01-01

Background Huntington's disease (HD) is an inherited neurodegenerative disorder triggered by an expanded polyglutamine tract in huntingtin that is thought to confer a new conformational property on this large protein. The propensity of small amino-terminal fragments with mutant, but not wild-type, glutamine tracts to self-aggregate is consistent with an altered conformation but such fragments occur relatively late in the disease process in human patients and mouse models expressing full-length mutant protein. This suggests that the altered conformational property may act within the full-length mutant huntingtin to initially trigger pathogenesis. Indeed, genotype-phenotype studies in HD have defined genetic criteria for the disease initiating mechanism, and these are all fulfilled by phenotypes associated with expression of full-length mutant huntingtin, but not amino-terminal fragment, in mouse models. As the in vitro aggregation of amino-terminal mutant huntingtin fragment offers a ready assay to identify small compounds that interfere with the conformation of the polyglutamine tract, we have identified a number of aggregation inhibitors, and tested whether these are also capable of reversing a phenotype caused by endogenous expression of mutant huntingtin in a striatal cell line from the HdhQ111/Q111 knock-in mouse. Results We screened the NINDS Custom Collection of 1,040 FDA approved drugs and bioactive compounds for their ability to prevent in vitro aggregation of Q58-htn 1–171 amino terminal fragment. Ten compounds were identified that inhibited aggregation with IC50 < 15 μM, including gossypol, gambogic acid, juglone, celastrol, sanguinarine and anthralin. Of these, both juglone and celastrol were effective in reversing the abnormal cellular localization of full-length mutant huntingtin observed in mutant HdhQ111/Q111 striatal cells. Conclusions At least some compounds identified as aggregation inhibitors also prevent a neuronal cellular phenotype caused by full-length mutant huntingtin, suggesting that in vitro fragment aggregation can act as a proxy for monitoring the disease-producing conformational property in HD. Thus, identification and testing of compounds that alter in vitro aggregation is a viable approach for defining potential therapeutic compounds that may act on the deleterious conformational property of full-length mutant huntingtin. PMID:15649316

Expression, purification, and characterization of recombinant human and murine milk fat globule-epidermal growth factor-factor 8.

PubMed

Castellanos, Erick R; Ciferri, Claudio; Phung, Wilson; Sandoval, Wendy; Matsumoto, Marissa L

2016-08-01

Milk fat globule-epidermal growth factor-factor 8 (MFG-E8), as its name suggests, is a major glycoprotein component of milk fat globules secreted by the mammary epithelium. Although its role in milk fat production is unclear, MFG-E8 has been shown to act as a bridge linking apoptotic cells to phagocytes for removal of these dying cells. MFG-E8 is capable of bridging these two very different cell types via interactions through both its epidermal growth factor (EGF)-like domain(s) and its lectin-type C domains. The EGF-like domain interacts with αVβ3 and αVβ5 integrins on the surface of phagocytes, whereas the C domains bind phosphatidylserine found on the surface of apoptotic cells. In an attempt to purify full-length, recombinant MFG-E8 expressed in either insect cells or CHO cells, we find that it is highly aggregated. Systematic truncation of the domain architecture of MFG-E8 indicates that the C domains are mainly responsible for the aggregation propensity. Addition of Triton X-100 to the conditioned cell culture media allowed partial recovery of non-aggregated, full-length MFG-E8. A more comprehensive detergent screen identified CHAPS as a stabilizer of MFG-E8 and allowed purification of a significant portion of non-aggregated, full-length protein. The CHAPS-stabilized recombinant MFG-E8 retained its natural ability to bind both αVβ3 and αVβ5 integrins and phosphatidylserine suggesting that it is properly folded and active. Herein we describe an efficient purification method for production of non-aggregated, full-length MFG-E8. Copyright © 2016 Elsevier Inc. All rights reserved.

Long-Term Results of Cartilage Repair after Allogeneic Transplantation of Cartilaginous Aggregates Formed from Bone Marrow-Derived Cells for Large Osteochondral Defects in Rabbit Knees.

PubMed

Yoshioka, Tomokazu; Mishima, Hajime; Sakai, Shinsuke; Uemura, Toshimasa

2013-10-01

The purpose of this study was to evaluate the long-term results of cartilage repair after allogeneic transplantation of cartilaginous aggregates formed from bone marrow-derived cells. Bone marrow cells were harvested from 12-day-old rabbits. The cells were subjected to a monolayer culture, and the spindle-shaped cells attached to the flask surface were defined as bone marrow-derived mesenchymal cells. After the monolayer culture, a 3-dimensional cartilaginous aggregate was formed using a bioreactor with chondrogenesis. We created osteochondral defects, measuring 5 mm in diameter and 4 mm in depth, at the femoral trochlea of 10-week-old rabbits. Two groups were established, the transplanted group in which the cartilaginous aggregate was transplanted into the defect, and the control group in which the defect was left untreated. Twenty-six and 52 weeks after surgery, the rabbits were sacrificed and their tissue repair status was evaluated macroscopically (International Cartilage Repair Society [ICRS] score) and histologically (O'Driscoll score). The ICRS scores were as follows: at week 26, 7.2 ± 0.5 and 7.6 ± 0.8; at week 52, 7.6 ± 1.1 and 9.7 ± 0.7, for the transplanted and control groups, respectively. O'Driscoll scores were as follows: at week 26, 12.6 ± 1.9 and 10.1 ± 1.9; at week 52, 9.6 ± 3.0 and 14.0 ± 1.4, each for transplanted and control groups, respectively. No significant differences were observed between the groups. This study demonstrates that allogeneic transplantation of cartilaginous aggregates formed from bone marrow-derived cells produces comparable long-term results based on macroscopic and histological outcome measures when compared with osteochondral defects that are left untreated.

Colonization Pattern of the Biocontrol Strain Pseudomonas chlororaphis MA 342 on Barley Seeds Visualized by Using Green Fluorescent Protein

PubMed Central

Tombolini, Riccardo; van der Gaag, Dirk Jan; Gerhardson, Berndt; Jansson, Janet K.

1999-01-01

Pseudomonas chlororaphis MA 342 is a potent biocontrol agent that can be used against several seed-borne diseases of cereal crops, including net blotch of barley caused by the fungus Drechslera teres. In this study, strain MA 342 was tagged with the gfp gene (encoding the green fluorescent protein) in order to study the fate of cells after seed inoculation. The gfp-tagged strain, MA 342G2, had the same biocontrol efficacy as the wild type when it was applied at high cell concentrations to seeds but was less effective at lower cell concentrations. By comparing cell counts determined by microscopy to the number of CFU, we found that the number of culturable cells was significantly lower than the total number of bacteria on seeds which were inoculated and dried for 20 h. Confocal microscopy and epifluorescence stereomicroscopy were used to determine the pattern of MA 342G2 colonization and cell aggregation on barley seeds. Immediately after inoculation of seeds, bacteria were found mainly under the seed glume, and there was no particular aggregation pattern. However, after the seeds were sown, irregularly distributed areas of bacterial aggregation were found, which reflected epiphytic colonization of glume cells. There was a trend towards bacterial aggregation near the embryo but never within the embryo. Bacterial aggregates were regularly found in the groove of each seed formed by the base of the coleoptile and the scutellum. Based on these results, we suggest that MA 342 colocalizes with the pathogen D. teres, which facilitates the action of the fungistatic compound(s) produced by this strain. PMID:10427065

Hsp70 and Antifibrillogenic Peptides Promote Degradation and Inhibit Intracellular Aggregation of Amyloidogenic Light Chains

PubMed Central

Dul, Jeanne L.; Davis, David P.; Williamson, Edward K.; Stevens, Fred J.; Argon, Yair

2001-01-01

In light chain (LC) amyloidosis an immunoglobulin LC assembles into fibrils that are deposited in various tissues. Little is known about how these fibrils form in vivo. We previously showed that a known amyloidogenic LC, SMA, can give rise to amyloid fibrils in vitro when a segment of one of its β sheets undergoes a conformational change, exposing an Hsp70 binding site. To examine SMA aggregation in vivo, we expressed it and its wild-type counterpart, LEN, in COS cells. While LEN is rapidly oxidized and subsequently secreted, newly synthesized SMA remains in the reduced state. Most SMA molecules are dislocated out of the ER into the cytosol, where they are ubiquitinylated and degraded by proteasomes. A parallel pathway for molecules that are not degraded is condensation into perinuclear aggresomes that are surrounded by vimentin-containing intermediate filaments and are dependent upon intact microtubules. Inhibition of proteasome activity shifts the balance toward aggresome formation. Intracellular aggregation is decreased and targeting to proteasomes improved by overexpression of the cytosolic chaperone Hsp70. Importantly, transduction into the cell of an Hsp70 target peptide, derived from the LC sequence, also reduces aggresome formation and increases SMA degradation. These results demonstrate that an amyloidogenic LC can aggregate intracellularly despite the common presentation of extracellular aggregates, and that a similar molecular surface mediates both in vitro fibril formation and in vivo aggregation. Furthermore, rationally designed peptides can be used to suppress this aggregation and may provide a feasible therapeutic approach. PMID:11266462

Hsp70 and antifibrillogenic peptides promote degradation and inhibit intracellular aggregation of amyloidogenic light chains.

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

Dul, J. L.; Davis, D. P.; Williamson, E. K.

2001-02-19

In light chain (LC) amyloidosis an immunoglobulin LC assembles into fibrils that are deposited in various tissues. Little is known about how these fibrils form in vivo. We previously showed that a known amyloidogenic LC, SMA, can give rise to amyloid fibrils in vitro when a segment of one of its {beta} sheets undergoes a conformational change, exposing an Hsp70 binding site. To examine SMA aggregation in vivo, we expressed it and its wild-type counterpart, LEN, in COS cells. While LEN is rapidly oxidized and subsequently secreted, newly synthesized SMA remains in the reduced state. Most SMA molecules are dislocatedmore » out of the ER into the cytosol, where they are ubiquitinylated and degraded by proteasomes. A parallel pathway for molecules that are not degraded is condensation into perinuclear aggresomes that are surrounded by vimentin-containing intermediate filaments and are dependent upon intact microtubules. Inhibition of proteasome activity shifts the balance toward aggresome formation. Intracellular aggregation is decreased and targeting to proteasomes improved by overexpression of the cytosolic chaperone Hsp70. Importantly, transduction into the cell of an Hsp70 target peptide, derived from the LC sequence, also reduces aggresome formation and increases SMA degradation. These results demonstrate that an amyloidogenic LC can aggregate intracellularly despite the common presentation of extracellular aggregates, and that a similar molecular surface mediates both in vitro fibril formation and in vivo aggregation. Furthermore, rationally designed peptides can be used to suppress this aggregation and may provide a feasible therapeutic approach.« less

Directional reversals enable Myxococcus xanthus cells to produce collective one-dimensional streams during fruiting-body formation

PubMed Central

Thutupalli, Shashi; Sun, Mingzhai; Bunyak, Filiz; Palaniappan, Kannappan; Shaevitz, Joshua W.

2015-01-01

The formation of a collectively moving group benefits individuals within a population in a variety of ways. The surface-dwelling bacterium Myxococcus xanthus forms dynamic collective groups both to feed on prey and to aggregate during times of starvation. The latter behaviour, termed fruiting-body formation, involves a complex, coordinated series of density changes that ultimately lead to three-dimensional aggregates comprising hundreds of thousands of cells and spores. How a loose, two-dimensional sheet of motile cells produces a fixed aggregate has remained a mystery as current models of aggregation are either inconsistent with experimental data or ultimately predict unstable structures that do not remain fixed in space. Here, we use high-resolution microscopy and computer vision software to spatio-temporally track the motion of thousands of individuals during the initial stages of fruiting-body formation. We find that cells undergo a phase transition from exploratory flocking, in which unstable cell groups move rapidly and coherently over long distances, to a reversal-mediated localization into one-dimensional growing streams that are inherently stable in space. These observations identify a new phase of active collective behaviour and answer a long-standing open question in Myxococcus development by describing how motile cell groups can remain statistically fixed in a spatial location. PMID:26246416

Sequential Coating of Insulin Secreting Beta Cells within Multilayers of Polysaccharide Nanogels.

PubMed

Bal, Tugba; Oran, Dilem Ceren; Sasaki, Yoshihiro; Akiyoshi, Kazunari; Kizilel, Seda

2018-05-01

Pancreatic islet transplantation has emerged as a promising treatment for type-1 diabetes (T1D); however, its clinical application is still limited by the life-long use of immunosuppressive drugs, insufficient number of islets to achieve normoglycemia, and large transplantation volume. This paper reports a unique approach for nanothin coating of insulin secreting beta cell aggregates. The coating is based on hydrophobic and covalent interactions between natural acrylate modified cholesterol bearing pullulan (CHPOA) nanogels and MIN6 beta cell aggregates. Beta cell aggregates are prepared as spheroids through hanging drop method, which is optimized with respect to hanging drop volume and initial number of beta cells. These aggregates, defined as pseudoislets, are coated with sequential layers of nanogels and are evaluated as viable and functional for insulin secretion. Coating experiments are carried out using physiologically compatible medium, where pseudoislets are not brought in contact with toxic prepolymer solutions used in existing approaches. This study offers new opportunities through coating of islets with advanced functional materials under completely physiological conditions for clinical translation of cell transplantation technology. The technique developed here will establish a new paradigm for creating tolerable grafts for other chronic diseases such as anemia, cancer, central nervous system (CNS) diseases. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Real-time amyloid aggregation monitoring with a photonic crystal-based approach.

PubMed

Santi, Sara; Musi, Valeria; Descrovi, Emiliano; Paeder, Vincent; Di Francesco, Joab; Hvozdara, Lubos; van der Wal, Peter; Lashuel, Hilal A; Pastore, Annalisa; Neier, Reinhard; Herzig, Hans Peter

2013-10-21

We propose the application of a new label-free optical technique based on photonic nanostructures to real-time monitor the amyloid-beta 1-42 (Aβ(1-42)) fibrillization, including the early stages of the aggregation process, which are related to the onset of the Alzheimer's Disease (AD). The aggregation of Aβ peptides into amyloid fibrils has commonly been associated with neuronal death, which culminates in the clinical features of the incurable degenerative AD. Recent studies revealed that cell toxicity is determined by the formation of soluble oligomeric forms of Aβ peptides in the early stages of aggregation. At this phase, classical amyloid detection techniques lack in sensitivity. Upon a chemical passivation of the sensing surface by means of polyethylene glycol, the proposed approach allows an accurate, real-time monitoring of the refractive index variation of the solution, wherein Aβ(1-42) peptides are aggregating. This measurement is directly related to the aggregation state of the peptide throughout oligomerization and subsequent fibrillization. Our findings open new perspectives in the understanding of the dynamics of amyloid formation, and validate this approach as a new and powerful method to screen aggregation at early stages. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Nano-aggregates: emerging delivery tools for tumor therapy.

PubMed

Sharma, Vinod Kumar; Jain, Ankit; Soni, Vandana

2013-01-01

A plethora of formulation techniques have been reported in the literature for site-specific targeting of water-soluble and -insoluble anticancer drugs. Along with other vesicular and particulate carrier systems, nano-aggregates have recently emerged as a novel supramolecular colloidal carrier with promise for using poorly water-soluble drugs in molecular targeted therapies. Nano-aggregates possess some inherent properties such as size in the nanometers, high loading efficiency, and in vivo stability. Nano-aggregates can provide site-specific drug delivery via either a passive or active targeting mechanism. Nano-aggregates are formed from a polymer-drug conjugated amphiphilic block copolymer. They are suitable for encapsulation of poorly water-soluble drugs by covalent conjugation as well as physical encapsulation. Because of physical encapsulation, a maximum amount of drug can be loaded in nano-aggregates, which helps to achieve a sufficiently high drug concentration at the target site. Active transport can be achieved by conjugating a drug with vectors or ligands that bind specifically to receptors being overexpressed in the tumor cells. In this review, we explore synthesis and tumor targeting potential of nano-aggregates with active and passive mechanisms, and we discuss various characterization parameters, ex vivo studies, biodistribution studies, clinical trials, and patents.

Descriptive parameters of the erythrocyte aggregation phenomenon using a laser transmission optical chip

NASA Astrophysics Data System (ADS)

Toderi, Martín A.; Castellini, Horacio V.; Riquelme, Bibiana D.

2017-01-01

The study of red blood cell (RBC) aggregation is of great interest because of its implications for human health. Altered RBC aggregation can lead to microcirculatory problems as in vascular pathologies, such as hypertension and diabetes, due to a decrease in the erythrocyte surface electric charge and an increase in the ligands present in plasma. The process of erythrocyte aggregation was studied in stasis situation (free shear stresses), using an optical chip based on the laser transmission technique. Kinetic curves of erythrocyte aggregation under different conditions were obtained, allowing evaluation and characterization of this process. Two main characteristics of blood that influence erythrocyte aggregation were analyzed: the erythrocyte surface anionic charge (EAC) after digestion with the enzyme trypsin and plasmatic protein concentration in suspension medium using plasma dissolutions in physiological saline with human albumin. A theoretical approach was evaluated to obtain aggregation and disaggregation ratios by syllectograms data fitting. Sensible parameters (Amp100, t) regarding a reduced erythrocyte EAC were determined, and other parameters (AI, M-Index) resulted that are representative of a variation in the plasmatic protein content of the suspension medium. These results are very useful for further applications in biomedicine.

Aberrant adhesion impacts early development in a Dictyostelium model for juvenile neuronal ceroid lipofuscinosis

PubMed Central

Huber, Robert J.; Myre, Michael A.; Cotman, Susan L.

2017-01-01

ABSTRACT Neuronal ceroid lipofuscinosis (NCL), also known as Batten disease, refers to a group of severe neurodegenerative disorders that primarily affect children. The most common subtype of the disease is caused by loss-of-function mutations in CLN3, which is conserved across model species from yeast to human. The precise function of the CLN3 protein is not known, which has made targeted therapy development challenging. In the social amoeba Dictyostelium discoideum, loss of Cln3 causes aberrant mid-to-late stage multicellular development. In this study, we show that Cln3-deficiency causes aberrant adhesion and aggregation during the early stages of Dictyostelium development. cln3− cells form ∼30% more multicellular aggregates that are comparatively smaller than those formed by wild-type cells. Loss of Cln3 delays aggregation, but has no significant effect on cell speed or cAMP-mediated chemotaxis. The aberrant aggregation of cln3− cells cannot be corrected by manually pulsing cells with cAMP. Moreover, there are no significant differences between wild-type and cln3− cells in the expression of genes linked to cAMP chemotaxis (e.g., adenylyl cyclase, acaA; the cAMP receptor, carA; cAMP phosphodiesterase, pdsA; g-protein α 9 subunit, gpaI). However, during this time in development, cln3− cells show reduced cell-substrate and cell-cell adhesion, which correlate with changes in the levels of the cell adhesion proteins CadA and CsaA. Specifically, loss of Cln3 decreases the intracellular level of CsaA and increases the amount of soluble CadA in conditioned media. Together, these results suggest that the aberrant aggregation of cln3− cells is due to reduced adhesion during the early stages of development. Revealing the molecular basis underlying this phenotype may provide fresh new insight into CLN3 function. PMID:27669405

Deposition or not? The fate of volcanic ash after aggregation processes

NASA Astrophysics Data System (ADS)

Mueller, Sebastian B.; Kueppers, Ulrich; Wadsworth, Fabian B.; Ayris, Paul M.; Casas, Ana S.; Cimarelli, Corrado; Ametsbichler, Jonathan; Delmelle, Pierre; Taddeucci, Jacopo; Jacob, Michael; Dingwell, Donald B.

2017-04-01

In the course of explosive volcanic eruptions, large amounts of ash are released into the atmosphere and may subsequently pose a threat to infrastructure, such as aviation industry. Ash plume forecasting is therefore a crucial tool for volcanic hazard mitigation but may be significantly affected by aggregation, altering the aerodynamic properties of particles. Models struggle with the implementation of aggregation since external conditions promoting aggregation have not been completely understood; in a previous study we have shown the rapid generation of ash aggregates through liquid bonding via the use of fluidization bed technology and further defined humidity and temperature ranges necessary to trigger aggregation. Salt (NaCl) was required for the recovery of stable aggregates, acting as a cementation agent and granting aggregate cohesion. A numerical model was used to explain the physics behind particle aggregation mechanisms and further predicted a dependency of aggregation efficiency on liquid binder viscosity. In this study we proof the effect of viscosity on particle aggregation. HCl and H2SO4 solutions were diluted to various concentrations resulting in viscosities between 1 and 2 mPas. Phonolitic and rhyolitic ash samples as well as soda-lime glass beads (serving as analogue material) were fluidized in the ProCell Lab® of Glatt Ingenieurtechnik GmbH and treated with the acids via a bottom-spray technique. Chemically driven interaction between acid liquids and surfaces of the three used materials led to crystal precipitation. Salt crystals (e.g. NaCl) have been confirmed through scanning electron microscopy (SEM) and leachate analysis. Both volcanic ash samples as well as the glass beads showed a clear dependency of aggregation efficiency on viscosity of the sprayed HCl solution. Spraying H2SO4 provoked a collapse of the fluidized bed and no aggregation has been observed. This is accounted by the high hygroscopicity of H2SO4. Dissolving CaCl2 (known to be a highly hygroscopic salt) in de-ionized water yielded comparable results without observable aggregation. In case of successful aggregation, concentration of salts has been found to be in the range of published values. We conclude that non-hygroscopic salt crystal precipitation from an aqueous liquid interacting with the glass phase in volcanic ash is a very efficient way to produce cohesive ash aggregates that can survive external forces acting during transport and sedimentation. Our parameterization of ash aggregation processes shall now be implemented in ash plume dispersal modelling for improved and more accurate ash distribution forecasting in the event of explosive volcanic eruptions.

HEMORHEOLOGICAL IMPLICATIONS OF PERFLUOROCARBON BASED OXYGEN CARRIER INTERACTION WITH COLLOID PLASMA EXPANDERS AND BLOOD

PubMed Central

Vásquez, Diana M.; Ortiz, Daniel; Alvarez, Oscar A.; Briceño, Juan C.; Cabrales, Pedro

2013-01-01

Perfluorocarbon (PFC) emulsion based oxygen carriers lack colloid osmotic pressure (COP) and must be administered with colloid-based plasma expanders (PEs). Although PFC emulsions have been widely studied, there is limited information about PFC emulsion interaction with PEs and blood. Their interaction forms aggregates due to electrostatic and rheological phenomena, and change blood rheology and blood flow. This study analyzes the effects of the interaction between PFC emulsions with blood in the presence of clinically-used PEs. The rheological behavior of the mixtures was analyzed in parallel with in vivo analysis of blood flow in microvessels using intravital microscopy when administered in a clinically relevant scenario. The interaction between the PFC emulsion and PE with blood produced PFC droplets and red blood cell (RBCs) aggregation, and increased blood viscosity. The PFC droplets formed aggregates when mixed with PEs containing electrolytes, and the aggregation increased with the electrolyte concentration. Mixtures of PFC with PEs that produced PFC aggregates also induced RCBs aggregation when mixed with blood, increasing blood viscosity at low shear rates. The more viscous suspension at low shear rates produced a blunted blood flow velocity profile in vivo relative to non-aggregating mixtures of PFC and PEs. For the PEs evaluated, albumin produced minimal to undetectable aggregation. PFC and PEs interaction with blood can affect sections of the microcirculation with low shear rate (e.g. arterioles, venules, and pulmonary circulation) because aggregates could cause capillary occlusion, decrease perfusion, pulmonary emboli, or focal ischemia. PMID:23606592

Force Sensitivity in Saccharomyces cerevisiae Flocculins.

PubMed

Chan, Cho X J; El-Kirat-Chatel, Sofiane; Joseph, Ivor G; Jackson, Desmond N; Ramsook, Caleen B; Dufrêne, Yves F; Lipke, Peter N

2016-01-01

Many fungal adhesins have short, β-aggregation-prone sequences that play important functional roles, and in the Candida albicans adhesin Als5p, these sequences cluster the adhesins after exposure to shear force. Here, we report that Saccharomyces cerevisiae flocculins Flo11p and Flo1p have similar β-aggregation-prone sequences and are similarly stimulated by shear force, despite being nonhomologous. Shear from vortex mixing induced the formation of small flocs in cells expressing either adhesin. After the addition of Ca(2+), yeast cells from vortex-sheared populations showed greatly enhanced flocculation and displayed more pronounced thioflavin-bright surface nanodomains. At high concentrations, amyloidophilic dyes inhibited Flo1p- and Flo11p-mediated agar invasion and the shear-induced increase in flocculation. Consistent with these results, atomic force microscopy of Flo11p showed successive force-distance peaks characteristic of sequentially unfolding tandem repeat domains, like Flo1p and Als5p. Flo11p-expressing cells bound together through homophilic interactions with adhesion forces of up to 700 pN and rupture lengths of up to 600 nm. These results are consistent with the potentiation of yeast flocculation by shear-induced formation of high-avidity domains of clustered adhesins at the cell surface, similar to the activation of Candida albicans adhesin Als5p. Thus, yeast adhesins from three independent gene families use similar force-dependent interactions to drive cell adhesion. IMPORTANCE The Saccharomyces cerevisiae flocculins mediate the formation of cellular aggregates and biofilm-like mats, useful in clearing yeast from fermentations. An important property of fungal adhesion proteins, including flocculins, is the ability to form catch bonds, i.e., bonds that strengthen under tension. This strengthening is based, at least in part, on increased avidity of binding due to clustering of adhesins in cell surface nanodomains. This clustering depends on amyloid-like β-aggregation of short amino acid sequences in the adhesins. In Candida albicans adhesin Als5, shear stress from vortex mixing can unfold part of the protein to expose aggregation-prone sequences, and then adhesins aggregate into nanodomains. We therefore tested whether shear stress from mixing can increase flocculation activity by potentiating similar protein remodeling and aggregation in the flocculins. The results demonstrate the applicability of the Als adhesin model and provide a rational framework for the enhancement or inhibition of flocculation in industrial applications.

Phthalocyanine tetrasulfonates affect the amyloid formation and cytotoxicity of alpha-synuclein.

PubMed

Lee, Eui-Nam; Cho, Hyun-Ju; Lee, Choong-Hwan; Lee, Daekyun; Chung, Kwang Chul; Paik, Seung R

2004-03-30

Alpha-synuclein is a pathological component of Parkinson's disease by constituting the filamentous component of Lewy bodies. Phthalocyanine (Pc) effects on the amyloidosis of alpha-synuclein have been examined. The copper complex of phthalocyanine tetrasulfonate (PcTS-Cu(2+)) caused the self-oligomerization of alpha-synuclein while Pc-Cu(2+) did not affect the protein, indicating that introduction of the sulfonate groups was critical for the selective protein interaction. The PcTS-Cu(2+) interaction with alpha-synuclein has occurred predominantly at the N-terminal region of the protein with a K(d) of 0.83 microM apart from the hydrophobic NAC (non-Abeta component of Alzheimer's disease amyloid) segment. Phthalocyanine tetrasulfonate (PcTS) lacking the intercalated copper ion also showed a considerable affinity toward alpha-synuclein with a K(d) of 3.12 microM, and its binding site, on the other hand, was located at the acidic C-terminus. These mutually exclusive interactions between PcTS and PcTS-Cu(2+) toward alpha-synuclein resulted in distinctive features on the kinetics of protein aggregation, morphologies of the final aggregates, and their in vitro cytotoxicities. The PcTS actually suppressed the fibrous amyloid formation of alpha-synuclein, but it produced the chopped-wood-looking protein aggregates. The aggregates showed rather low toxicity (9.5%) on human neuroblastoma cells (SH-SY5Y). In fact, the PcTS was shown to effectively rescue the cell death of alpha-synuclein overexpressing cells caused by the lactacystin treatment as a proteasome inhibitor. The anti-aggregative and anti-amyloidogenic properties of PcTS were also demonstrated with alcohol dehydrogenase, glutathione S-transferase, and amyloid beta/A4 protein under their aggregative conditions. The PcTS-Cu(2+), on the other hand, promoted the protein aggregation of alpha-synuclein, which gave rise to the fibrillar protein aggregates whose cytotoxicity became significant to 35.8%. Taken together, the data provided in this study indicate that PcTS/PcTS-Cu(2+) could be considered as possible candidates for the development of therapeutic or prophylactic strategies against the alpha-synuclein-related neurodegenerative disorders.

Anthraquinones inhibit tau aggregation and dissolve Alzheimer's paired helical filaments in vitro and in cells.

PubMed

Pickhardt, Marcus; Gazova, Zuzana; von Bergen, Martin; Khlistunova, Inna; Wang, Yipeng; Hascher, Antje; Mandelkow, Eva-Maria; Biernat, Jacek; Mandelkow, Eckhard

2005-02-04

The abnormal aggregation of tau protein into paired helical filaments (PHFs) is one of the hallmarks of Alzheimer's disease. Aggregation takes place in the cytoplasm and could therefore be cytotoxic for neurons. To find inhibitors of PHF aggregation we screened a library of 200,000 compounds. The hits found in the PHF inhibition assay were also tested for their ability to dissolve preformed PHFs. The results were obtained using a thioflavin S fluorescence assay for the detection and quantification of tau aggregation in solution, a tryptophan fluorescence assay using tryptophan-containing mutants of tau, and confirmed by a pelleting assay and electron microscopy of the products. Here we demonstrate the feasibility of the approach with several compounds from the family of anthraquinones, including emodin, daunorubicin, adriamycin, and others. They were able to inhibit PHF formation with IC50 values of 1-5 microm and to disassemble preformed PHFs at DC50 values of 2-4 microm. The compounds had a similar activity for PHFs made from different tau isoforms and constructs. The compounds did not interfere with the stabilization of microtubules by tau. Tau-inducible neuroblastoma cells showed the formation of tau aggregates and concomitant cytotoxicity, which could be prevented by inhibitors. Thus, small molecule inhibitors could provide a basis for the development of tools for the treatment of tau pathology in AD and other tauopathies.

Cytoplasmic Location of α1A Voltage-Gated Calcium Channel C-Terminal Fragment (Cav2.1-CTF) Aggregate Is Sufficient to Cause Cell Death

PubMed Central

Takahashi, Makoto; Obayashi, Masato; Ishiguro, Taro; Sato, Nozomu; Niimi, Yusuke; Ozaki, Kokoro; Mogushi, Kaoru; Mahmut, Yasen; Tanaka, Hiroshi; Tsuruta, Fuminori; Dolmetsch, Ricardo; Yamada, Mitsunori; Takahashi, Hitoshi; Kato, Takeo; Mori, Osamu; Eishi, Yoshinobu; Mizusawa, Hidehiro; Ishikawa, Kinya

2013-01-01

The human α1A voltage-dependent calcium channel (Cav2.1) is a pore-forming essential subunit embedded in the plasma membrane. Its cytoplasmic carboxyl(C)-tail contains a small poly-glutamine (Q) tract, whose length is normally 4∼19 Q, but when expanded up to 20∼33Q, the tract causes an autosomal-dominant neurodegenerative disorder, spinocerebellar ataxia type 6 (SCA6). A recent study has shown that a 75-kDa C-terminal fragment (CTF) containing the polyQ tract remains soluble in normal brains, but becomes insoluble mainly in the cytoplasm with additional localization to the nuclei of human SCA6 Purkinje cells. However, the mechanism by which the CTF aggregation leads to neurodegeneration is completely elusive, particularly whether the CTF exerts more toxicity in the nucleus or in the cytoplasm. We tagged recombinant (r)CTF with either nuclear-localization or nuclear-export signal, created doxycyclin-inducible rat pheochromocytoma (PC12) cell lines, and found that the CTF is more toxic in the cytoplasm than in the nucleus, the observations being more obvious with Q28 (disease range) than with Q13 (normal-length). Surprisingly, the CTF aggregates co-localized both with cAMP response element-binding protein (CREB) and phosphorylated-CREB (p-CREB) in the cytoplasm, and Western blot analysis showed that the quantity of CREB and p-CREB were both decreased in the nucleus when the rCTF formed aggregates in the cytoplasm. In human brains, polyQ aggregates also co-localized with CREB in the cytoplasm of SCA6 Purkinje cells, but not in other conditions. Collectively, the cytoplasmic Cav2.1-CTF aggregates are sufficient to cause cell death, and one of the pathogenic mechanisms may be abnormal CREB trafficking in the cytoplasm and reduced CREB and p-CREB levels in the nuclei. PMID:23505410

Role of the H-bond between L53 and T56 for Aquaporin-4 epitope in Neuromyelitis Optica.

PubMed

Pisani, Francesco; Simone, Laura; Gargano, Concetta Domenica; De Bellis, Manuela; Cibelli, Antonio; Mola, Maria Grazia; Catacchio, Giacomo; Frigeri, Antonio; Svelto, Maria; Nicchia, Grazia Paola

2017-03-01

Aquaporin-4 (AQP4) is the CNS water channel organized into well-ordered protein aggregates called Orthogonal Arrays of Particles (OAPs). Neuromyelitis Optica (NMO) is an autoimmune disease caused by anti-OAP autoantibodies (AQP4-IgG). Molecular Dynamics (MD) simulations have identified an H-bond between L53 and T56 as the key for AQP4 epitope and therefore of potential interest for drug design in NMO field. In the present study, we have experimentally tested this MD-prediction using the classic mutagenesis approach. We substituted T56 with V56 and tested this mutant for AQP4 aggregates and AQP4-IgG binding. gSTED super-resolution microscopy showed that the mutation does not affect AQP4 aggregate dimension; immunofluorescence and cytofluorimetric analysis demonstrated its unaltered AQP4-IgG binding, therefore invalidating the MD-prediction. We later investigated whether AQP4, expressed in Sf9 insect and HEK-293F cells, is able to correctly aggregate before and after the purification steps usually applied to obtain AQP4 crystal. The results demonstrated that AQP4-IgG recognizes AQP4 expressed in Sf9 and HEK-293F cells by immunofluorescence even though BN-PAGE analysis showed that AQP4 forms smaller aggregates when expressed in insect cells compared to mammalian cell lines. Notably, after AQP4 purification, from both insect and HEK-293F cells, no aggregates are detectable by BN-PAGE and AQP4-IgG binding is impaired in sandwich ELISA assays. All together these results indicate that 1) the MD prediction under analysis is not supported by experimental data and 2) the procedure to obtain AQP4 crystals might affect its native architecture and, as a consequence, MD simulations. In conclusion, given the complex nature of the AQP4 epitope, MD might not be the suitable for molecular medicine advances in NMO. Copyright © 2016. Published by Elsevier B.V.

Tracking protein aggregation and mislocalization in cells with flow cytometry.

PubMed

Ramdzan, Yasmin M; Polling, Saskia; Chia, Cheryl P Z; Ng, Ivan H W; Ormsby, Angelique R; Croft, Nathan P; Purcell, Anthony W; Bogoyevitch, Marie A; Ng, Dominic C H; Gleeson, Paul A; Hatters, Danny M

2012-03-18

We applied pulse-shape analysis (PulSA) to monitor protein localization changes in mammalian cells by flow cytometry. PulSA enabled high-throughput tracking of protein aggregation, translocation from the cytoplasm to the nucleus and trafficking from the plasma membrane to the Golgi as well as stress-granule formation. Combining PulSA with tetracysteine-based oligomer sensors in a cell model of Huntington's disease enabled further separation of cells enriched with monomers, oligomers and inclusion bodies.

Surface Mn(II) oxidation actuated by a multicopper oxidase in a soil bacterium leads to the formation of manganese oxide minerals

PubMed Central

Zhang, Zhen; Zhang, Zhongming; Chen, Hong; Liu, Jin; Liu, Chang; Ni, Hong; Zhao, Changsong; Ali, Muhammad; Liu, Fan; Li, Lin

2015-01-01

In this manuscript, we report that a bacterial multicopper oxidase (MCO266) catalyzes Mn(II) oxidation on the cell surface, resulting in the surface deposition of Mn(III) and Mn(IV) oxides and the gradual formation of bulky oxide aggregates. These aggregates serve as nucleation centers for the formation of Mn oxide micronodules and Mn-rich sediments. A soil-borne Escherichia coli with high Mn(II)-oxidizing activity formed Mn(III)/Mn(IV) oxide deposit layers and aggregates under laboratory culture conditions. We engineered MCO266 onto the cell surfaces of both an activity-negative recipient and wild-type strains. The results confirmed that MCO266 governs Mn(II) oxidation and initiates the formation of deposits and aggregates. By contrast, a cell-free substrate, heat-killed strains, and intracellularly expressed or purified MCO266 failed to catalyze Mn(II) oxidation. However, purified MCO266 exhibited Mn(II)-oxidizing activity when combined with cell outer membrane component (COMC) fractions in vitro. We demonstrated that Mn(II) oxidation and aggregate formation occurred through an oxygen-dependent biotic transformation process that requires a certain minimum Mn(II) concentration. We propose an approximate electron transfer pathway in which MCO266 transfers only one electron to convert Mn(II) to Mn(III) and then cooperates with other COMC electron transporters to transfer the other electron required to oxidize Mn(III) to Mn(IV). PMID:26039669

Attachment and biofilm formation by various serotypes of Salmonella as influenced by cellulose production and thin aggregative fimbriae biosynthesis.

PubMed

Jain, Sudeep; Chen, Jinru

2007-11-01

This study was undertaken to quantify thin aggregative fimbriae and cellulose produced by Salmonella and to evaluate their roles in attachment and biofilm formation on polystyrene and glass surfaces. Thin aggregative fimbriae and cellulose produced by four wild-type and two pairs of Salmonella, representing four different colony morphotypes (rdar: red, dry, and rough; pdar: pink, dry, and rough; bdar: brown, dry, and rough; and saw: smooth and white), were quantified. The ability of the Salmonella cells to attach and form biofilms on the selected surfaces was evaluated in Luria-Bertani (LB) broth with or without salt (0.5%) or glucose (2%) at 28 degrees C during a 7-day period. The cells expressing the rdar or pdar colony morphotypes produced significantly greater amounts of thin aggregative fimbriae and cellulose on LB no salt agar, respectively. The cells expressing the rdar colony morphotype attached in higher numbers and formed more biofilm than did the cells expressing the pdar colony morphotype. The members of the pairs expressing the bdar colony morphotype attached more efficiently and formed more biofilm on the tested surfaces than did their counterparts expressing the saw colony morphotype. These results indicated that thin aggregative fimbriae impart attachment ability to Salmonella and, upon coexpression with cellulose, enhance biofilm formation on certain abiotic surfaces. The knowledge acquired in the study may help develop better cleaning strategies for food processing equipment.

Surface Mn(II) oxidation actuated by a multicopper oxidase in a soil bacterium leads to the formation of manganese oxide minerals.

PubMed

Zhang, Zhen; Zhang, Zhongming; Chen, Hong; Liu, Jin; Liu, Chang; Ni, Hong; Zhao, Changsong; Ali, Muhammad; Liu, Fan; Li, Lin

2015-06-03

In this manuscript, we report that a bacterial multicopper oxidase (MCO266) catalyzes Mn(II) oxidation on the cell surface, resulting in the surface deposition of Mn(III) and Mn(IV) oxides and the gradual formation of bulky oxide aggregates. These aggregates serve as nucleation centers for the formation of Mn oxide micronodules and Mn-rich sediments. A soil-borne Escherichia coli with high Mn(II)-oxidizing activity formed Mn(III)/Mn(IV) oxide deposit layers and aggregates under laboratory culture conditions. We engineered MCO266 onto the cell surfaces of both an activity-negative recipient and wild-type strains. The results confirmed that MCO266 governs Mn(II) oxidation and initiates the formation of deposits and aggregates. By contrast, a cell-free substrate, heat-killed strains, and intracellularly expressed or purified MCO266 failed to catalyze Mn(II) oxidation. However, purified MCO266 exhibited Mn(II)-oxidizing activity when combined with cell outer membrane component (COMC) fractions in vitro. We demonstrated that Mn(II) oxidation and aggregate formation occurred through an oxygen-dependent biotic transformation process that requires a certain minimum Mn(II) concentration. We propose an approximate electron transfer pathway in which MCO266 transfers only one electron to convert Mn(II) to Mn(III) and then cooperates with other COMC electron transporters to transfer the other electron required to oxidize Mn(III) to Mn(IV).

The Myxococcus xanthus Spore Cuticula Protein C Is a Fragment of FibA, an Extracellular Metalloprotease Produced Exclusively in Aggregated Cells

PubMed Central

Lee, Bongsoo; Mann, Petra; Grover, Vidhi; Treuner-Lange, Anke; Kahnt, Jörg; Higgs, Penelope I.

2011-01-01

Myxococcus xanthus is a soil bacterium with a complex life cycle involving distinct cell fates, including production of environmentally resistant spores to withstand periods of nutrient limitation. Spores are surrounded by an apparently self-assembling cuticula containing at least Proteins S and C; the gene encoding Protein C is unknown. During analyses of cell heterogeneity in M. xanthus, we observed that Protein C accumulated exclusively in cells found in aggregates. Using mass spectrometry analysis of Protein C either isolated from spore cuticula or immunoprecipitated from aggregated cells, we demonstrate that Protein C is actually a proteolytic fragment of the previously identified but functionally elusive zinc metalloprotease, FibA. Subpopulation specific FibA accumulation is not due to transcriptional regulation suggesting post-transcriptional regulation mechanisms mediate its heterogeneous accumulation patterns. PMID:22174937

In vitro differentiation of mouse embryonic stem (mES) cells using the hanging drop method.

PubMed

Wang, Xiang; Yang, Phillip

2008-07-23

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, This promising of science is leading scientists to investigate the possibility of cell-based therapies to treat disease. When culture in suspension without antidifferentiation factors, embryonic stem cells spontaneously differentiate and form three-dimensional multicellular aggregates. These cell aggregates are called embryoid bodies(EB). Hanging drop culture is a widely used EB formation induction method. The rounded bottom of hanging drop allows the aggregation of ES cells which can provide mES cells a good environment for forming EBs. The number of ES cells aggregatied in a hanging drop can be controlled by varying the number of cells in the initial cell suspension to be hung as a drop from the lid of Petri dish. Using this method we can reproducibly form homogeneous EBs from a predetermined number of ES cells.

Comparison of sea turtle thrombocyte aggregation to human platelet aggregation in whole blood.

PubMed

Soslau, Gerald; Prest, Phillip J; Class, Reiner; George, Robert; Paladino, Frank; Violetta, Gary

2005-11-01

The endangered sea turtles are living "fossils" that afford us an opportunity to study the hemostatic process as it likely existed millions of years ago. There are essentially no data about turtle thrombocyte aggregation prior to our studies. Thrombocytes are nucleated cells that serve the same hemostatic functions as the anucleated mammalian platelet. Sea turtle thrombocytes aggregate in response to collagen and beta-thrombin. Ristocetin induces an agglutination/aggregation response indicating the presence of a von Willebrand-like receptor, GPIb, found in all mammalian platelets. Samples treated with alpha-thrombin plus gamma-thrombin followed by ristocetin results in a rapid, stronger response than ristocetin alone. These responses are inhibited by the RGDS peptide that blocks fibrinogen cross-linking of mammalian platelets via the fibrinogen receptor, GPIIb/IIIa. Three platelet-like proteins, GPIb, GPIIb/IIIa and P-selection are detected in sea turtle thrombocytes by fluorescence activated cell sorting. Turtle thrombocytes do not respond to ADP, epinephrine, serotonin, thromboxane A2 mimetic, U46619, trypsin, or alpha-thrombin and gamma-thrombin added alone. Comparison of hemostasis in sea turtles to other vertebrates could provide a framework for understanding the structure/function and evolution of these pathways and their individual components.

Extracellular Polymeric Substance Production and Aggregated Bacteria Colonization Influence the Competition of Microbes in Biofilms.

PubMed

Jayathilake, Pahala G; Jana, Saikat; Rushton, Steve; Swailes, David; Bridgens, Ben; Curtis, Tom; Chen, Jinju

2017-01-01

The production of extracellular polymeric substance (EPS) is important for the survival of biofilms. However, EPS production is costly for bacteria and the bacterial strains that produce EPS (EPS+) grow in the same environment as non-producers (EPS-) leading to competition between these strains for nutrients and space. The outcome of this competition is likely to be dependent on factors such as initial attachment, EPS production rate, ambient nutrient levels and quorum sensing. We use an Individual-based Model (IbM) to study the competition between EPS+ and EPS- strains by varying the nature of initial colonizers which can either be in the form of single cells or multicellular aggregates. The microbes with EPS+ characteristics obtain a competitive advantage if they initially colonize the surface as smaller aggregates and are widely spread-out between the cells of EPS-, when both are deposited on the substratum. Furthermore, the results show that quorum sensing-regulated EPS production may significantly reduce the fitness of EPS producers when they initially deposit as aggregates. The results provide insights into how the distribution of bacterial aggregates during initial colonization could be a deciding factor in the competition among different strains in biofilms.

Conformational analysis of misfolded protein aggregation by FRET and live-cell imaging techniques.

PubMed

Kitamura, Akira; Nagata, Kazuhiro; Kinjo, Masataka

2015-03-16

Cellular homeostasis is maintained by several types of protein machinery, including molecular chaperones and proteolysis systems. Dysregulation of the proteome disrupts homeostasis in cells, tissues, and the organism as a whole, and has been hypothesized to cause neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and Huntington's disease (HD). A hallmark of neurodegenerative disorders is formation of ubiquitin-positive inclusion bodies in neurons, suggesting that the aggregation process of misfolded proteins changes during disease progression. Hence, high-throughput determination of soluble oligomers during the aggregation process, as well as the conformation of sequestered proteins in inclusion bodies, is essential for elucidation of physiological regulation mechanism and drug discovery in this field. To elucidate the interaction, accumulation, and conformation of aggregation-prone proteins, in situ spectroscopic imaging techniques, such as Förster/fluorescence resonance energy transfer (FRET), fluorescence correlation spectroscopy (FCS), and bimolecular fluorescence complementation (BiFC) have been employed. Here, we summarize recent reports in which these techniques were applied to the analysis of aggregation-prone proteins (in particular their dimerization, interactions, and conformational changes), and describe several fluorescent indicators used for real-time observation of physiological states related to proteostasis.

Increased expression of p62/SQSTM1 in prion diseases and its association with pathogenic prion protein.

PubMed

Homma, Takujiro; Ishibashi, Daisuke; Nakagaki, Takehiro; Satoh, Katsuya; Sano, Kazunori; Atarashi, Ryuichiro; Nishida, Noriyuki

2014-03-28

Prion diseases are neurodegenerative disorders characterized by the aggregation of abnormally folded prion protein (PrP(Sc)). In this study, we focused on the mechanism of clearance of PrP(Sc), which remains unclear. p62 is a cytosolic protein known to mediate both the formation and degradation of aggregates of abnormal proteins. The levels of p62 protein increased in prion-infected brains and persistently infected cell cultures. Upon proteasome inhibition, p62 co-localized with PrP(Sc), forming a large aggregate in the perinuclear region, hereafter referred to as PrP(Sc)-aggresome. These aggregates were surrounded with autophagosome marker LC3 and lysosomes in prion-infected cells. Moreover, transient expression of the phosphomimic form of p62, which has enhanced ubiquitin-binding activity, reduced the amount of PrP(Sc) in prion-infected cells, indicating that the activation of p62 could accelerate the clearance of PrP(Sc). Our findings would thus suggest that p62 could be a target for the therapeutic control of prion diseases.

Zinc chlorophyll aggregates as hole transporters for biocompatible, natural-photosynthesis-inspired solar cells

NASA Astrophysics Data System (ADS)

Li, Yue; Sasaki, Shin-ichi; Tamiaki, Hitoshi; Liu, Cheng-Liang; Song, Jiaxing; Tian, Wenjing; Zheng, Enqiang; Wei, Yingjin; Chen, Gang; Fu, Xueqi; Wang, Xiao-Feng

2015-11-01

The intriguing properties of extremely efficient delocalization and migration of excitons in chlorophyll (Chl) J-type aggregates have inspired intense research activities toward their structural understanding, functional interpretation and mimicry synthesis. Herein, we demonstrated the J-aggregates of zinc methyl 3-devinyl-3-hydroxymethyl-pyropheophorbide a (ZnChl-1) generated by spin-coating method for the application as a hole transporter in titania-based solar cells using methyl trans-32-carboxypyropheophorbide a (H2Chl-2) or its zinc complex (ZnChl-2) as the sensitizer. The effective carrier mobility of the J-aggregates films was determined by the organic field-effect transistor to be 6.2 × 10-4 cm2 V-1 s-1. Solar cells sharing the architecture of FTO/H2Chl-2 or ZnChl-2 on TiO2/(ZnChl-1)n/Ag were fabricated and the factors that presumably determine their photovoltaic performances were discussed. The photovoltaic devices studied herein employing inexpensive and pollution-free biomaterials provide a unique solution of utilizing solar energy with a care of the important environmental issue.

Extract from Aronia melanocarpa fruits potentiates the inhibition of platelet aggregation in the presence of endothelial cells

PubMed Central

Luzak, Boguslawa; Golanski, Jacek; Rozalski, Marek; Krajewska, Urszula; Olas, Beata

2010-01-01

Introduction Some polyphenolic compounds extracted from Aronia melanocarpa fruits (AM) have been reported to be cardioprotective agents. In this study we evaluated the ability of AM extract to increase the efficacy of human umbilical vein endothelial cells (HUVECs) to inhibit platelet functions in vitro. Material and methods This study encompasses two models of monitoring platelet reactivity: optical aggregation and platelet degranulation (monitored as the surface CD62P expression) in PRP upon the stimulation with ADP. Results We observed that only at low concentrations (5 µg/ml) did AM extract significantly improve antiplatelet action of HUVECs towards ADP-activated platelets in the aggregation test. Conclusions It is concluded that the potentiating effect of AM extract on the endothelial cell-mediated inhibition of platelet aggregation clearly depends on the used concentrations of Aronia-derived active compounds. Therefore, despite these encouraging preliminary outcomes on the beneficial effects of AM extract polyphenols, more profound dose-effect studies should certainly be considered before the implementation of Aronia-originating compounds in antiplatelet therapy and the prevention of cardiovascular diseases. PMID:22371737

Engraftment of human induced pluripotent stem cell-derived hepatocytes in immunocompetent mice via 3D co-aggregation and encapsulation

PubMed Central

Song, Wei; Lu, Yen-Chun; Frankel, Angela S.; An, Duo; Schwartz, Robert E.; Ma, Minglin

2015-01-01

Cellular therapies for liver diseases and in vitro models for drug testing both require functional human hepatocytes (Hum-H), which have unfortunately been limited due to the paucity of donor liver tissues. Human pluripotent stem cells (hPSCs) represent a promising and potentially unlimited cell source to derive Hum-H. However, the hepatic functions of these hPSC-derived cells to date are not fully comparable to adult Hum-H and are more similar to fetal ones. In addition, it has been challenging to obtain functional hepatic engraftment of these cells with prior studies having been done in immunocompromised animals. In this report, we demonstrated successful engraftment of human induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (iPS-H) in immunocompetent mice by pre-engineering 3D cell co-aggregates with stromal cells (SCs) followed by encapsulation in recently developed biocompatible hydrogel capsules. Notably, upon transplantation, human albumin and α1-antitrypsin (A1AT) in mouse sera secreted by encapsulated iPS-H/SCs aggregates reached a level comparable to the primary Hum-H/SCs control. Further immunohistochemistry of human albumin in retrieved cell aggregates confirmed the survival and function of iPS-H. This proof-of-concept study provides a simple yet robust approach to improve the engraftment of iPS-H, and may be applicable to many stem cell-based therapies. PMID:26592180

Development of alginate-based aggregate inoculants of Methylobacterium sp. and Azospirillum brasilense tested under in vitro conditions to promote plant growth.

PubMed

Joe, M M; Saravanan, V S; Islam, M R; Sa, T

2014-02-01

To develop co-aggregated bacterial inoculant comprising of Methylobacterium oryzae CBMB20/Methylobacterium suomiense CBMB120 strains with Azospirillum brasilense (CW903) strain and testing their efficiency as inoculants for plant growth promotion (PGP). Biofilm formation and co-aggregation efficiency was studied between A. brasilense CW903 and methylobacterial strains M. oryzae CBMB20 and M. suomiense CBMB120. Survival and release of these co-aggregated bacterial strains entrapped in alginate beads were assessed. PGP attributes of the co-aggregated bacterial inoculant were tested in tomato plants under water-stressed conditions. Results suggest that the biofilm formation efficiency of the CBMB20 and CBMB120 strains increased by 15 and 34%, respectively, when co-cultivated with CW903. Co-aggregation with CW903 enhanced the survivability of CBMB20 strain in alginate beads. Water stress index score showed least stress index in plants inoculated with CW903 and CBMB20 strains maintained as a co-aggregated inoculant. This study reports the development of co-aggregated cell inoculants containing M. oryzae CBMB20 and A. brasilense CW903 strains conferred better shelf life and stress abatement in inoculated tomato plants. These findings could be extended to other PGP bacterial species to develop multigeneric bioinoculants with multiple benefits for various crops. © 2013 The Society for Applied Microbiology.

A passive physical model for DnaK chaperoning

NASA Astrophysics Data System (ADS)

Uhl, Lionel; Dumont, Audrey; Dukan, Sam

2018-03-01

Almost all living organisms use protein chaperones with a view to preventing proteins from misfolding or aggregation either spontaneously or during cellular stress. This work uses a reaction-diffusion stochastic model to describe the dynamic localization of the Hsp70 chaperone DnaK in Escherichia coli cells during transient proteotoxic collapse characterized by the accumulation of insoluble proteins. In the model, misfolded (‘abnormal’) proteins are produced during alcoholic stress and have the propensity to aggregate with a polymerization-like kinetics. When aggregates diffuse more slowly they grow larger. According to Michaelis-Menten-type kinetics, DnaK has the propensity to bind with misfolded proteins or aggregates in order to catalyse refolding. To match experimental fluorescence microscopy data showing clusters of DnaK-GFP localized in multiple foci, the model includes spatial zones with local reduced diffusion rates to generate spontaneous assemblies of DnaK called ‘foci’. Numerical simulations of our model succeed in reproducing the kinetics of DnaK localization experimentally observed. DnaK starts from foci, moves to large aggregates during acute stress, resolves those aggregates during recovery and finally returns to its initial punctate localization pattern. Finally, we compare real biological events with hypothetical repartitions of the protein aggregates or DnaK. We then notice that DnaK action is more efficient on protein aggregates than on protein homogeneously distributed.

SOD1 aggregation in astrocytes following ischemia/reperfusion injury: a role of NO-mediated S-nitrosylation of protein disulfide isomerase (PDI).

PubMed

Chen, Xueping; Guan, Teng; Li, Chen; Shang, Huifang; Cui, Liying; Li, Xin-Min; Kong, Jiming

2012-10-12

Ubiquitinated-protein aggregates are implicated in cerebral ischemia/reperfusion injury. The very presence of these ubiquitinated-protein aggregates is abnormal and seems to be disease-related. However, it is not clear what leads to aggregate formation and whether the aggregations represent a reaction to aggregate-mediated neurodegeneration. To study the nitrosative stress-induced protein aggregation in cerebral ischemia/reperfusion injury, we used primary astrocyte cultures as a cell model, and systematically examined their iNOS expression and consequent NO generation following oxygen glucose deprivation and reperfusion. The expression of protein disulfide isomerase (PDI) and copper-zinc superoxide dismutase (SOD1) were also examined, and the biochemical interaction between PDI and SOD1 was determined by immunoprecipitation. In addition, the levels of S-nitrosylated PDI in cultured astrocytes after oxygen glucose deprivation and reperfusion treatment were measured using the biotin-switch assay. The formation of ubiquitinated-protein aggregates was detected by immunoblot and immunofluorescence staining. Our data showed that the up-regulation of iNOS expression after oxygen glucose deprivation and reperfusion treatment led to excessive NO generation. Up-regulation of PDI and SOD1 was also identified in cultured astrocytes following oxygen glucose deprivation and reperfusion, and these two proteins were found to bind to each other. Furthermore, the increased nitrosative stress due to ischemia/reperfusion injury was highly associated with NO-induced S-nitrosylation of PDI, and this S-nitrosylation of PDI was correlated with the formation of ubiquitinated-protein aggregates; the levels of S-nitrosylated PDI increased in parallel with the formation of aggregates. When NO generation was pharmacologically inhibited by iNOS specific inhibitor 1400W, S-nitrosylation of PDI was significantly blocked. In addition, the formation of ubiquitinated-protein aggregates in cultured astrocytes following oxygen glucose deprivation and reperfusion was also suppressed by 1400W. Interestingly, these aggregates were colocalized with SOD1, which was found to co-immunoprecipitate with PDI. NO-mediated S-nitrosylation of PDI may be involved in the formation of the SOD1-linked ubiquitinated-protein aggregates in cerebral ischemia/reperfusion injury.

Interplay Between Protein Homeostasis Networks in Protein Aggregation and Proteotoxicity

PubMed Central

Douglas, Peter M.; Cyr, Douglas M.

2010-01-01

The misfolding and aggregation of disease proteins is characteristic of numerous neurodegenerative diseases. Particular neuronal populations are more vulnerable to proteotoxicity while others are more apt to tolerate the misfolding and aggregation of disease proteins. Thus, the cellular environment must play a significant role in determining whether disease proteins are converted into toxic or benign forms. The endomembrane network of eukaryotes divides the cell into different subcellular compartments that possess distinct sets of molecular chaperones and protein interaction networks. Chaperones act as agonists and antagonists of disease protein aggregation to prevent the accumulation of toxic intermediates in the aggregation pathway. Interacting partners can also modulate the conformation and localization of disease proteins and thereby influence proteotoxicity. Thus, interplay between these protein homeostasis network components can modulate the self-association of disease proteins and determine whether they elicit a toxic or benign outcome. PMID:19768782

Inhibition of p53 Mutant Peptide Aggregation In Vitro by Cationic Osmolyte Acetylcholine Chloride.

PubMed

Chen, Zhaolin; Kanapathipillai, Mathumai

2017-01-01

Mutations of tumor suppressor protein p53 are present in almost about 50% of all cancers. It has been reported that the p53 mutations cause aggregation and subsequent loss of p53 function, leading to cancer progression. Here in this study we focus on the inhibitory effects of cationic osmolyte molecules acetylcholine chloride, and choline on an aggregation prone 10 amino acid p53 mutant peptide WRPILTIITL, and the corresponding wildtype peptide RRPILTIITL in vitro. The characterization tools used for this study include Thioflavin- T (ThT) induced fluorescence, transmission electron microscopy (TEM), congo red binding, turbidity, dynamic light scattering (DLS), and cell viability assays. The results show that acetylcholine chloride in micromolar concentrations significantly inhibit p53 mutant peptide aggregation in vitro, and could be promising candidate for p53 mutant/ misfolded protein aggregation inhibition. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Concepts and tools to exploit the potential of bacterial inclusion bodies in protein science and biotechnology.

PubMed

Gatti-Lafranconi, Pietro; Natalello, Antonino; Ami, Diletta; Doglia, Silvia Maria; Lotti, Marina

2011-07-01

Cells have evolved complex and overlapping mechanisms to protect their proteins from aggregation. However, several reasons can cause the failure of such defences, among them mutations, stress conditions and high rates of protein synthesis, all common consequences of heterologous protein production. As a result, in the bacterial cytoplasm several recombinant proteins aggregate as insoluble inclusion bodies. The recent discovery that aggregated proteins can retain native-like conformation and biological activity has opened the way for a dramatic change in the means by which intracellular aggregation is approached and exploited. This paper summarizes recent studies towards the direct use of inclusion bodies in biotechnology and for the detection of bottlenecks in the folding pathways of specific proteins. We also review the major biophysical methods available for revealing fine structural details of aggregated proteins and which information can be obtained through these techniques. © 2011 The Authors Journal compilation © 2011 FEBS.

Aggregation behaviour and electron injection/recombination dynamics of symmetrical and unsymmetrical Zn-phthalocyanines on TiO2 film.

PubMed

Ashokkumar, R; Kathiravan, A; Ramamurthy, P

2014-01-21

We have synthesized symmetrical and unsymmetrical Zn-phthalocyanine derivatives (PZnPc, MPZnPc and TPZnPc) for dye sensitized solar cells (DSSCs). Steady state and time-resolved absorption and fluorescence studies were performed in DMF solvent and on a TiO2 surface. The mode and extent of aggregation (H- and J-aggregates) of ZnPc adsorbed on a TiO2 surface were demonstrated. MPZnPc shows both H- and J-aggregation, while TPZnPc shows only H-aggregation. Moreover, the fluorescence of ZnP/TiO2 was completely quenched and this was assigned to electron injection from excited ZnPc to TiO2. Energy level calculations show both ZnPc deriviatives have enough driving force to inject electrons into the conduction band of TiO2. Furthermore, the radical cation of ZnPc was observed in nanosecond transient absorption measurements.

Three-photon-excited luminescence from unsymmetrical cyanostilbene aggregates: morphology tuning and targeted bioimaging.

PubMed

Mandal, Amal Kumar; Sreejith, Sivaramapanicker; He, Tingchao; Maji, Swarup Kumar; Wang, Xiao-Jun; Ong, Shi Li; Joseph, James; Sun, Handong; Zhao, Yanli

2015-05-26

We report an experimental observation of aggregation-induced enhanced luminescence upon three-photon excitation in aggregates formed from a class of unsymmetrical cyanostilbene derivatives. Changing side chains (-CH3, -C6H13, -C7H15O3, and folic acid) attached to the cyanostilbene core leads to instantaneous formation of aggregates with sizes ranging from micrometer to nanometer scale in aqueous conditions. The crystal structure of a derivative with a methyl side chain reveals the planarization in the unsymmetrical cyanostilbene core, causing luminescence from corresponding aggregates upon three-photon excitation. Furthermore, folic acid attached cyanostilbene forms well-dispersed spherical nanoaggregates that show a high three-photon cross-section of 6.0 × 10(-80) cm(6) s(2) photon(-2) and high luminescence quantum yield in water. In order to demonstrate the targeted bioimaging capability of the nanoaggregates, three cell lines (HEK293 healthy cell line, MCF7 cancerous cell line, and HeLa cancerous cell line) were employed for the investigations on the basis of their different folate receptor expression level. Two kinds of nanoaggregates with and without the folic acid targeting ligand were chosen for three-photon bioimaging studies. The cell viability of three types of cells incubated with high concentration of nanoaggregates still remained above 70% after 24 h. It was observed that the nanoaggregates without the folic acid unit could not undergo the endocytosis by both healthy and cancerous cell lines. No obvious endocytosis of folic acid attached nanoaggregates was observed from the HEK293 and MCF7 cell lines having a low expression of the folate receptor. Interestingly, a significant amount of endocytosis and internalization of folic acid attached nanoaggregates was observed from HeLa cells with a high expression of the folate receptor under three-photon excitation, indicating targeted bioimaging of folic acid attached nanoaggregates to the cancer cell line. This study presents a paradigm of using organic nanoaggregates for targeted three-photon bioimaging.

Dielectric aggregation kinetics of cells in a uniform AC electric field.

PubMed

Tada, Shigeru; Natsuya, Tomoyuki; Tsukamoto, Akira

2014-01-01

Cell manipulation and separation technologies have potential biological and medical applications, including advanced clinical protocols such as tissue engineering. An aggregation model was developed for a human carcinoma (HeLa) cell suspension exposed to a uniform AC electric field, in order to explore the field-induced structure formation and kinetics of cell aggregates. The momentum equations of cells under the action of the dipole-dipole interaction were solved theoretically and the total time required to form linear string-like cluster was derived. The results were compared with those of a numerical simulation. Experiments using HeLa cells were also performed for comparison. The total time required to form linear string-like clusters was derived from a simple theoretical model of the cell cluster kinetics. The growth rates of the average string length of cell aggregates showed good agreement with those of the numerical simulation. In the experiment, cells were found to form massive clusters on the bottom of a chamber. The results imply that the string-like cluster grows rapidly by longitudinal attraction when the electric field is first applied and that this process slows at later times and is replaced by lateral coagulation of short strings. The findings presented here are expected to enable design of methods for the organization of three-dimensional (3D) cellular structures without the use of micro-fabricated substrates, such as 3D biopolymer scaffolds, to manipulate cells into spatial arrangement.

Large scale isolation, growth, and function of porcine neonatal islet cells.

PubMed Central

Korbutt, G S; Elliott, J F; Ao, Z; Smith, D K; Warnock, G L; Rajotte, R V

1996-01-01

Based upon existing methods of isolating fetal porcine islet tissue, a simple, reliable procedure was developed for the preparation of porcine neonatal islet cell aggregates with a reproducible and defined cellular composition. After 9 d of in vitro culture, tissue from one neonatal pig pancreas yielded approximately 50,000 islet cell aggregates, consisting of primarily epithelial cells (57%) and pancreatic endocrine cells (35%). During the culture period, the total beta cell mass decreased initially, but subsequently increased 1.5-fold between days 3 and 9. Transplantation of grafts consisting of 3 x 10(5) beta cells (1,000 aggregated) under the kidney capsule of alloxan-diabetic nude mice corrected hyperglycemia in 75% (10/13) of the animals, whereas, 100% (20/20) of recipients implanted with 6 x 10(5) beta cells (2,000 aggregates) achieved euglycemia within 8 wk posttransplantation. Nephrectomy of the graft bearing kidney at 14 wk posttransplantation resulted in hyperglycemia in all recipients, and examination of the grafts revealed the presence of numerous well-granulated insulin- and glucagon-containing cells. The cellular insulin content of these grafts was 20 to 30-fold higher than at the time of transplantation. These results indicate that the neonatal porcine pancrease can be used as a source of large numbers of viable islet cells, which have the potential for growth both in vitro and in vivo, and exhibit the metabolic capacity to correct diabetes in nude mice. PMID:8621802

The association of peroxisomes with the developing cell plate in dividing onion root cells depends on actin microfilaments and myosin.

PubMed

Collings, David A; Harper, John D I; Vaughn, Kevin C

2003-12-01

We have investigated changes in the distribution of peroxisomes through the cell cycle in onion ( Allium cepa L.) root meristem cells with immunofluorescence and electron microscopy, and in leek ( Allium porrum L.) epidermal cells with immunofluorescence and peroxisomal-targeted green fluorescent protein. During interphase and mitosis, peroxisomes distribute randomly throughout the cytoplasm, but beginning late in anaphase, they accumulate at the division plane. Initially, peroxisomes occur within the microtubule phragmoplast in two zones on either side of the developing cell plate. However, as the phragmoplast expands outwards to form an annulus, peroxisomes redistribute into a ring immediately inside the location of the microtubules. Peroxisome aggregation depends on actin microfilaments and myosin. Peroxisomes first accumulate in the division plane prior to the formation of the microtubule phragmoplast, and throughout cytokinesis, always co-localise with microfilaments. Microfilament-disrupting drugs (cytochalasin and latrunculin), and a putative inhibitor of myosin (2,3-butanedione monoxime), inhibit aggregation. We propose that aggregated peroxisomes function in the formation of the cell plate, either by regulating hydrogen peroxide production within the developing cell plate, or by their involvement in recycling of excess membranes from secretory vesicles via the beta-oxidation pathway. Differences in aggregation, a phenomenon which occurs in onion, some other monocots and to a lesser extent in tobacco BY-2 suspension cells, but which is not obvious in the roots of Arabidopsis thaliana (L.) Heynh., may reflect differences within the primary cell walls of these plants.

The S/T-Rich Motif in the DNAJB6 Chaperone Delays Polyglutamine Aggregation and the Onset of Disease in a Mouse Model.

PubMed

Kakkar, Vaishali; Månsson, Cecilia; de Mattos, Eduardo P; Bergink, Steven; van der Zwaag, Marianne; van Waarde, Maria A W H; Kloosterhuis, Niels J; Melki, Ronald; van Cruchten, Remco T P; Al-Karadaghi, Salam; Arosio, Paolo; Dobson, Christopher M; Knowles, Tuomas P J; Bates, Gillian P; van Deursen, Jan M; Linse, Sara; van de Sluis, Bart; Emanuelsson, Cecilia; Kampinga, Harm H

2016-04-21

Expanded CAG repeats lead to debilitating neurodegenerative disorders characterized by aggregation of proteins with expanded polyglutamine (polyQ) tracts. The mechanism of aggregation involves primary and secondary nucleation steps. We show how a noncanonical member of the DNAJ-chaperone family, DNAJB6, inhibits the conversion of soluble polyQ peptides into amyloid fibrils, in particular by suppressing primary nucleation. This inhibition is mediated by a serine/threonine-rich region that provides an array of surface-exposed hydroxyl groups that bind to polyQ peptides and may disrupt the formation of the H bonds essential for the stability of amyloid fibrils. Early prevention of polyQ aggregation by DNAJB6 occurs also in cells and leads to delayed neurite retraction even before aggregates are visible. In a mouse model, brain-specific coexpression of DNAJB6 delays polyQ aggregation, relieves symptoms, and prolongs lifespan, pointing to DNAJB6 as a potential target for disease therapy and tool for unraveling early events in the onset of polyQ diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

Induction of IAPP amyloid deposition and associated diabetic abnormalities by a prion-like mechanism

PubMed Central

Morales-Scheihing, Diego; Salvadores, Natalia; Moreno-Gonzalez, Ines; Gonzalez, Cesar; Shahnawaz, Mohammad

2017-01-01

Although a large proportion of patients with type 2 diabetes (T2D) accumulate misfolded aggregates composed of the islet amyloid polypeptide (IAPP), its role in the disease is unknown. Here, we show that pancreatic IAPP aggregates can promote the misfolding and aggregation of endogenous IAPP in islet cultures obtained from transgenic mouse or healthy human pancreas. Islet homogenates immunodepleted with anti-IAPP–specific antibodies were not able to induce IAPP aggregation. Importantly, intraperitoneal inoculation of pancreatic homogenates containing IAPP aggregates into transgenic mice expressing human IAPP dramatically accelerates IAPP amyloid deposition, which was accompanied by clinical abnormalities typical of T2D, including hyperglycemia, impaired glucose tolerance, and a substantial reduction on β cell number and mass. Finally, induction of IAPP deposition and diabetic abnormalities were also induced in vivo by administration of IAPP aggregates prepared in vitro using pure, synthetic IAPP. Our findings suggest that some of the pathologic and clinical alterations of T2D might be transmissible through a similar mechanism by which prions propagate in prion diseases. PMID:28765400

Genome-wide RNA interference screen identifies previously undescribed regulators of polyglutamine aggregation

PubMed Central

Nollen, Ellen A. A.; Garcia, Susana M.; van Haaften, Gijs; Kim, Soojin; Chavez, Alejandro; Morimoto, Richard I.; Plasterk, Ronald H. A.

2004-01-01

Protein misfolding and the formation of aggregates are increasingly recognized components of the pathology of human genetic disease and hallmarks of many neurodegenerative disorders. As exemplified by polyglutamine diseases, the propensity for protein misfolding is associated with the length of polyglutamine expansions and age-dependent changes in protein-folding homeostasis, suggesting a critical role for a protein homeostatic buffer. To identify the complement of protein factors that protects cells against the formation of protein aggregates, we tested transgenic Caenorhabditis elegans strains expressing polyglutamine expansion yellow fluorescent protein fusion proteins at the threshold length associated with the age-dependent appearance of protein aggregation. We used genome-wide RNA interference to identify genes that, when suppressed, resulted in the premature appearance of protein aggregates. Our screen identified 186 genes corresponding to five principal classes of polyglutamine regulators: genes involved in RNA metabolism, protein synthesis, protein folding, and protein degradation; and those involved in protein trafficking. We propose that each of these classes represents a molecular machine collectively comprising the protein homeostatic buffer that responds to the expression of damaged proteins to prevent their misfolding and aggregation. PMID:15084750

Estimation of Scatterer Diameter by Normalized Power Spectrum of High-Frequency Ultrasonic RF Echo for Assessment of Red Blood Cell Aggregation

NASA Astrophysics Data System (ADS)

Fukushima, Taku; Hasegawa, Hideyuki; Kanai, Hiroshi

2011-07-01

Red blood cell (RBC) aggregation, as one of the determinants of blood viscosity, plays an important role in blood rheology, including the condition of blood. RBC aggregation is induced by the adhesion of RBCs when the electrostatic repulsion between RBCs weakens owing to increases in protein and saturated fatty acid levels in blood, excessive RBC aggregation leads to various circulatory diseases. This study was conducted to establish a noninvasive quantitative method for assessment of RBC aggregation. The power spectrum of ultrasonic RF echoes from nonaggregating RBCs, which shows the frequency property of scattering, exhibits Rayleigh behavior. On the other hand, ultrasonic RF echoes from aggregating RBCs contain the components of reflection, which have no frequency dependence. By dividing the measured power spectrum of echoes from RBCs in the lumen by that of echoes from a posterior wall of the vein in the dorsum manus, the attenuation property of the propagating medium and the frequency responses of transmitting and receiving transducers are removed from the former spectrum. RBC aggregation was assessed by the diameter of a scatterer, which was estimated by minimizing the square difference between the measured normalized power spectrum and the theoretical power spectrum. In this study, spherical scatterers with diameters of 5, 11, 15, and 30 µm were measured in basic experiments. The estimated scatterer diameters were close to the actual diameters. Furthermore, the transient change of the scatterer diameters were measured in an in vivo experiment with respect to a 24-year-old healthy male during the avascularization using a cuff. The estimated diameters (12-22 µm) of RBCs during avascularization were larger than the diameters (4-8 µm) at rest and after recirculation. These results show the possibility of the use of the proposed method for noninvasive assessment of RBC aggregation.

Is trehalose an autophagic inducer? Unraveling the roles of non-reducing disaccharides on autophagic flux and alpha-synuclein aggregation.

PubMed

Yoon, Ye-Seul; Cho, Eun-Duk; Jung Ahn, Woo; Won Lee, Kyung; Lee, Seung-Jae; Lee, He-Jin

2017-10-05

Autophagy is a pivotal intracellular process by which cellular macromolecules are degraded upon various stimuli. A failure in the degradation of autophagic substrates such as impaired organelles and protein aggregates leads to their accumulations, which are characteristics of many neurodegenerative diseases. Pharmacological activation of autophagy has thus been considered a prospective therapeutic approach for treating neurodegenerative diseases. Among a number of autophagy-inducing agents, trehalose has received attention for its beneficial effects in different disease models of neurodegeneration. However, how trehalose promotes autophagy has not been fully revealed. We investigated the influence of trehalose and other disaccharides upon autophagic flux and aggregation of α-synuclein, a protein linked to Parkinson's disease. In differentiated human neuroblastoma and primary rat cortical neuron culture models, treatment with trehalose and other disaccharides resulted in accumulation of lipidated LC3 (LC3-II), p62, and autophagosomes, whereas it decreased autolysosomes. On the other hand, addition of Bafilomycin A1 to trehalose treatments had relatively marginal effect, an indicative of autophagic flux blockage. In concordance with these results, the cells treated with trehalose exhibited an incremental tendency in α-synuclein aggregation. Secretion of α-synuclein was also elevated in the culture medium upon trehalose treatment, thereby significantly increasing intercellular transmission of this protein. Despite the substantial increase in α-synuclein aggregation, which normally leads to cell death, cell viability was not affected upon treatment with trehalose, suggesting an autophagy-independent protective function of trehalose against protein aggregates. This study demonstrates that, although trehalose has been widely considered an autophagic inducer, it may be actually a potent blocker of the autophagic flux.

Early alterations of red blood cell rheology in critically ill patients.

PubMed

Reggiori, Giulia; Occhipinti, Giovanna; De Gasperi, Andrea; Vincent, Jean-Louis; Piagnerelli, Michael

2009-12-01

To investigate red blood cell rheology in a large intensive care unit population on admission, and to assess the possible influence of comorbidities on the rheology. : Prospective study. Medico-surgical intensive care unit with 31 beds. All intensive care unit admissions during a 5-month period and 20 healthy volunteers. Blood sampling. A total of 196 intensive care patients (160 without and 36 with sepsis) and 20 healthy volunteers were studied. Red blood cell rheology (deformability and aggregation) was assessed ex vivo using the laser-assisted optical rotational cell analyzer (LORCA; Mechatronics Instruments BV, AN Zwaag, Netherlands) within the first 24 hrs after intensive care unit admission. Red blood cell deformability was determined by the elongation index in relation to the shear stress (0.3 to 50 Pa) applied on the red blood cell membrane surface. Aggregation was assessed by the aggregation index. Septic patients were more likely to have anemia, coagulation abnormalities, and comorbidities than were nonseptic patients. Red blood cell deformability was significantly altered in septic compared to nonseptic patients and volunteers for the majority of shear stress rates studied. The aggregation index was greater in septic patients than in volunteers (67.9% [54.7-73.5] vs. 61.8% [58.2-68.4]; p < .05). Only sepsis and hematologic disease influenced the elongation index (both p < .01). Other comorbidities, like cancer, diabetes mellitus, cirrhosis, and terminal renal failure, had no effect on the elongation index. Aggregation index was related to the degree of organ failure (Sequential Organ Failure Assessment score), the red blood cell count, and fibrinogen concentrations. Early alterations of red blood cell rheology are common in intensive care unit patients, especially in those with sepsis. Comorbidities (other than hematologic diseases) do not significantly influence these abnormalities. These alterations could contribute to the microcirculatory alterations observed in critically ill patients.

Heterogeneity of antibody response to human platelet transfusion.

PubMed Central

Wu, K K; Thompson, J S; Koepke, J A; Hoak, J C; Flink, R

1976-01-01

To study the antibody response to human platelet transfusions, nine thrombocytopenia patients with bone marrow failure were given 6 U (3X10(11)) of random platelet concentrates twice a week. Before transfusion, none of the patients had preexisting antibodies detectable with lymphocytotoxicity, platelet aggregation, or capillary leukoagglutination techniques. After receiving 18-78 U of platelets, they became refractory to further transfusions of random platelets and alloantibodies were detectable. Two patterns of antibody response could be identified. In three patients, the sera were not lymphocytotoxic with a panel of standard cells in which all the known HLA antigens in the first and second series were represented at least once. Yet, they caused platelet aggregation with 30, 24, and 60%, respectively, of a donor population studied. The aggregating activities were inhibited by antihuman IgG but not by antihuman IgA or antihuman IgM antiserum. The aggregating antibodies could be absorbed out with donor platelets but not lymphocytes or granulocytes. Antibodies from two of these patients aggregated platelets of their respective siblings matched for both HLA haplotypes. Transfusion of platelets from these two siblings did not increase the platelet count while platelets obtained from aggregation-negative donors did. The sera from the remaining six patients were lymphocytotoxic with 15-100% of the panel of standard cells. They also had aggregating antibodies, which could be absorbed out by both platelets and lymphocytes, suggesting that they were HLA antibodies. These data suggest that the development of platelet-specific antibodies may play an important role in the immunological rejection of isologous platelets, and should be considered in the selection of donors for patients who are refractory to platelets from random donors. PMID:956376

Changes in translation rate modulate stress-induced damage of diverse proteins

PubMed Central

Kim, Heejung

2013-01-01

Proteostasis is the maintenance of the proper function of cellular proteins. Hypertonic stress disrupts proteostasis and causes rapid and widespread protein aggregation and misfolding in the nematode Caenorhabditis elegans. Optimal survival in hypertonic environments requires degradation of damaged proteins. Inhibition of protein synthesis occurs in response to diverse environmental stressors and may function in part to minimize stress-induced protein damage. We recently tested this idea directly and demonstrated that translation inhibition by acute exposure to cycloheximide suppresses hypertonicity-induced aggregation of polyglutamine::YFP (Q35::YFP) in body wall muscle cells. In this article, we further characterized the relationship between protein synthesis and hypertonic stress-induced protein damage. We demonstrate that inhibition of translation reduces hypertonic stress-induced formation and growth of Q35::YFP, Q44::YFP, and α-synuclein aggregates; misfolding of paramyosin and ras GTPase; and aggregation of multiple endogenous proteins expressed in diverse cell types. Activation of general control nonderepressible-2 (GCN-2) kinase signaling during hypertonic stress inhibits protein synthesis via phosphorylation of eukaryotic initiation factor-2α (eIF-2α). Inhibition of GCN-2 activation prevents the reduction in translation rate and greatly exacerbates the formation and growth of Q35::YFP aggregates and the aggregation of endogenous proteins. The current studies together with our previous work provide the first direct demonstration that hypertonic stress-induced reduction in protein synthesis minimizes protein aggregation and misfolding. Reduction in translation rate also serves as a signal that activates osmoprotective gene expression. The cellular proteostasis network thus plays a critical role in minimizing hypertonic stress-induced protein damage, in degrading stress-damaged proteins, and in cellular osmosensing and signaling. PMID:24153430

Controlled Aggregation of Ferritin to Modulate MRI Relaxivity

PubMed Central

Bennett, Kevin M.; Shapiro, Erik M.; Sotak, Christopher H.; Koretsky, Alan P.

2008-01-01

Ferritin is an iron storage protein expressed in varying concentrations in mammalian cells. The deposition of ferric iron in the core of ferritin makes it a magnetic resonance imaging contrast agent, and ferritin has recently been proposed as a gene expression reporter protein for magnetic resonance imaging. To date, ferritin has been overexpressed in vivo and has been coexpressed with transferrin receptor to increase iron loading in cells. However, ferritin has a relatively low T2 relaxivity (R2 ≈ 1 mM−1s−1) at typical magnetic field strengths and so requires high levels of expression to be detected. One way to modulate the transverse relaxivity of a superparamagnetic agent is to cause it to aggregate, thereby manipulating the magnetic field gradients through which water diffuses. In this work, it is demonstrated by computer simulation and in vitro that aggregation of ferritin can alter relaxivity. The effects of aggregate size and intraaggregate perturber spacing on R2 are studied. Computer modeling indicates that the optimal spacing of the ferritin molecules in aggregate for increasing R2 is 100–200 nm for a typical range of water diffusion rates. Chemical cross-linking of ferritin at 12 Å spacing led to a 70% increase in R2 compared to uncross-linked ferritin controls. To modulate ferritin aggregation in a potentially biologically relevant manner, ferritin was attached to actin and polymerized in vitro. The polymerization of ferritin-F-actin caused a 20% increase in R2 compared to unpolymerized ferritin-G-actin. The R2-value was increased by another 10% by spacing the ferritin farther apart on the actin filaments. The modulation of ferritin aggregation by binding to cytoskeletal elements may be a useful strategy to make a functional reporter gene for magnetic resonance imaging. PMID:18326661

Intraglomerular thrombotic tendency and glomerular ADPase. Unilateral impairment of ADPase elicits a proaggregatory microenvironment in experimental glomerulonephritis

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

Poelstra, K.; Baller, J.F.; Hardonk, M.J.

1991-04-01

It has been proposed, predominantly from ex vivo studies, that glomerular ADPase may function as an antithrombotic principle within the rat kidney. Therefore, intraglomerular platelet aggregation was studied in vivo in rats after impairment of glomerular ADPase activity using local X-irradiation (20 Gy). Biochemical assays in suspensions of glomeruli obtained from rats 24 hours after local X-irradiation (group I) demonstrated a significant reduction in ADPase activity as compared to sham treated rats (group II; p less than 0.01). Cytochemical observations at the ultrastructural level showed that this reduction in glomerular enzyme activity represents in particular ADPase activity detectable in themore » basement membrane. Following X-irradiation, intraglomerular platelet aggregation was quantitatively studied in two groups of rats. Both groups received X-irradiation of the left kidney (20 Gy). Twenty-four hours after X-irradiation, animals received an intravenous injection of either 0.5 ml of saline (group III; N = 6) or 0.5 ml of heterologous nephrotoxic serum (NTS; group IV; N = 6). Subsequently, 24 hours after this injection, platelet aggregation in left kidneys was compared with aggregation in contralateral non-X-irradiated kidneys. The results showed that while X-irradiation per se did not induce intraglomerular platelet aggregation as compared with the contralateral kidney (0.20 +/- 0.08% versus 0.17 +/- 0.06% platelet aggregation/glomerulus), a significant increase in platelet aggregation could be demonstrated in X-irradiated kidneys in the early phase of nephrotoxic serum nephritis as compared with the contralateral nephritic kidney. A potential effect of altered influx of inflammatory cells after X-irradiation could be excluded since no difference in H2O2 producing cells was observed between left and right kidneys.« less

Insights into the Aggregation Mechanism of PolyQ Proteins with Different Glutamine Repeat Lengths.

PubMed

Yushchenko, Tetyana; Deuerling, Elke; Hauser, Karin

2018-04-24

Polyglutamine (polyQ) diseases, including Huntington's disease, result from the aggregation of an abnormally expanded polyQ repeat in the affected protein. The length of the polyQ repeat is essential for the disease's onset; however, the molecular mechanism of polyQ aggregation is still poorly understood. Controlled conditions and initiation of the aggregation process are prerequisites for the detection of transient intermediate states. We present an attenuated total reflection Fourier-transform infrared spectroscopic approach combined with protein immobilization to study polyQ aggregation dependent on the polyQ length. PolyQ proteins were engineered mimicking the mammalian N-terminus fragment of the Huntingtin protein and containing a polyQ sequence with the number of glutamines below (Q11), close to (Q38), and above (Q56) the disease threshold. A monolayer of the polyQ construct was chemically immobilized on the internal reflection element of the attenuated total reflection cell, and the aggregation was initiated via enzymatic cleavage. Structural changes of the polyQ sequence were monitored by time-resolved infrared difference spectroscopy. We observed faster aggregation kinetics for the longer sequences, and furthermore, we could distinguish β-structured intermediates for the different constructs, allowing us to propose aggregation mechanisms dependent on the repeat length. Q11 forms a β-structured aggregate by intermolecular interaction of stretched monomers, whereas Q38 and Q56 undergo conformational changes to various β-structured intermediates, including intramolecular β-sheets. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

In vitro spatially organizing the differentiation in individual multicellular stem cell aggregates.

PubMed

Qi, Hao; Huang, Guoyou; Han, Yu Long; Lin, Wang; Li, Xiujun; Wang, Shuqi; Lu, Tian Jian; Xu, Feng

2016-01-01

With significant potential as a robust source to produce specific somatic cells for regenerative medicine, stem cells have attracted increasing attention from both academia and government. In vivo, stem cell differentiation is a process under complicated regulations to precisely build tissue with unique spatial structures. Since multicellular spheroidal aggregates of stem cells, commonly called as embryoid bodies (EBs), are considered to be capable of recapitulating the events in early stage of embryonic development, a variety of methods have been developed to form EBs in vitro for studying differentiation of embryonic stem cells. The regulation of stem cell differentiation is crucial in directing stem cells to build tissue with the correct spatial architecture for specific functions. However, stem cells within the three-dimensional multicellular aggregates undergo differentiation in a less unpredictable and spatially controlled manner in vitro than in vivo. Recently, various microengineering technologies have been developed to manipulate stem cells in vitro in a spatially controlled manner. Herein, we take the spotlight on these technologies and researches that bring us the new potential for manipulation of stem cells for specific purposes.

Intrinsically disordered proteins aggregate at fungal cell-to-cell channels and regulate intercellular connectivity.

PubMed

Lai, Julian; Koh, Chuan Hock; Tjota, Monika; Pieuchot, Laurent; Raman, Vignesh; Chandrababu, Karthik Balakrishna; Yang, Daiwen; Wong, Limsoon; Jedd, Gregory

2012-09-25

Like animals and plants, multicellular fungi possess cell-to-cell channels (septal pores) that allow intercellular communication and transport. Here, using a combination of MS of Woronin body-associated proteins and a bioinformatics approach that identifies related proteins based on composition and character, we identify 17 septal pore-associated (SPA) proteins that localize to the septal pore in rings and pore-centered foci. SPA proteins are not homologous at the primary sequence level but share overall physical properties with intrinsically disordered proteins. Some SPA proteins form aggregates at the septal pore, and in vitro assembly assays suggest aggregation through a nonamyloidal mechanism involving mainly α-helical and disordered structures. SPA loss-of-function phenotypes include excessive septation, septal pore degeneration, and uncontrolled Woronin body activation. Together, our data identify the septal pore as a complex subcellular compartment and focal point for the assembly of unstructured proteins controlling diverse aspects of intercellular connectivity.

Neuroglian-positive plasmatocytes of Manduca sexta and the initiation of hemocyte attachment to foreign surfaces.

PubMed

Nardi, James B; Pilas, Barbara; Bee, Charles Mark; Zhuang, Shufei; Garsha, Karl; Kanost, Michael R

2006-01-01

Observations of hemocyte aggregation on abiotic surfaces suggested that certain plasmatocytes from larvae of Manduca sexta act as foci for hemocyte aggregation. To establish how these particular plasmatocytes form initial attachments to foreign surfaces, they were cultured separately from other selected populations of hemocytes. While all circulating plasmatocytes immunolabel with anti-beta-integrin monoclonal antibody (MAb), only these larger plasmatocytes immunolabel with a MAb to the adhesion protein neuroglian. Neuroglian-negative plasmatocytes and granular cells that have been magnetically segregated from the majority of granular cells adhere to each other but fail to adhere to foreign substrata; by contrast, neuroglian-positive plasmatocytes that segregate with most granular cells adhere firmly to a substratum. Hemocytes form stable aggregates around the large, neuroglian-positive plasmatocytes. However, if neuroglian-positive plasmatocytes are separated from most granular cells, attachment of these plasmatocytes to foreign surfaces is suppressed.

Selection against BALB/c strain cells in mouse chimaeras

PubMed Central

Tang, Pin-Chi; MacKay, Gillian E.; Flockhart, Jean H.; Keighren, Margaret A.; Kopakaki, Anna

2018-01-01

ABSTRACT It has been shown previously that BALB/c strain embryos tend to contribute poorly to mouse aggregation chimaeras. In the present study we showed that BALB/c cells were not preferentially allocated to any extraembryonic lineages of mouse aggregation chimaeras, but their contribution decreased during the early postimplantation period and they were significantly depleted by E8.5. The development of BALB/c strain preimplantation embryos lagged behind embryos from some other strains and the contribution that BALB/c and other embryos made to chimaeras correlated with their developmental stage at E2.5. This relationship suggests that the poor contribution of BALB/c embryos to aggregation chimaeras is at least partly a consequence of generalised selection related to slow or delayed preimplantation development. The suitability of BALB/c embryos for maximising the ES cell contribution to mouse ES cell chimaeras is also discussed. PMID:29330350

Differential Function of N-Cadherin and Cadherin-7 in the Control of Embryonic Cell Motility

PubMed Central

Dufour, Sylvie; Beauvais-Jouneau, Alice; Delouvée, Annie; Thiery, Jean Paul

1999-01-01

Similar amounts of N-cadherin and cadherin-7, the prototypes of type I and type II cadherin, induced cell-cell adhesion in murine sarcoma 180 transfectants, Ncad-1 and cad7-29, respectively. However, in the initial phase of aggregation, Ncad-1 cells aggregated more rapidly than cad7-29 cells. Isolated Ncad-1 and cad7-29 cells adhered and spread in a similar manner on fibronectin (FN), whereas aggregated cad7-29 cells were more motile and dispersed than aggregated Ncad-1 cells. cad7-29 cells established transient contacts with their neighbors which were stabilized if FN-cell interactions were perturbed. In contrast, Ncad-1 cells remained in close contact when they migrated on FN. Both β-catenin and cadherin were more rapidly downregulated in cad7-29 than in Ncad-1 cells treated with cycloheximide, suggesting a higher turnover rate for cadherin-7–mediated cell-cell contacts than for those mediated by N-cadherin. The extent of FN-dependent focal adhesion kinase phosphorylation was much lower if the cells had initiated N-cadherin–mediated rather than cadherin-7–mediated cell adhesion before plating. On grafting into the embryo, Ncad-1 cells did not migrate and remained at or close to the graft site, even after 48 h, whereas grafted cad7-29 cells dispersed efficiently into embryonic structures. Thus, the adhesive phenotype of cadherin-7–expressing cells is regulated by the nature of the extracellular matrix environment which also controls the migratory behavior of the cells. In addition, adhesions mediated by different cadherins differentially regulate FN-dependent signaling. The transient contacts specifically observed in cadherin- 7–expressing cells may also be important in the control of cell motility. PMID:10427101

Protein Aggregates and Novel Presenilin Gene Variants in Idiopathic Dilated Cardiomyopathy

PubMed Central

Gianni, Davide; Li, Airong; Tesco, Giuseppina; McKay, Kenneth M.; Moore, John; Raygor, Kunal; Rota, Marcello; Gwathmey, Judith K; Dec, G William; Aretz, Thomas; Leri, Annarosa; Semigran, Marc J; Anversa, Piero; Macgillivray, Thomas E; Tanzi, Rudolph E.; Monte, Federica del

2010-01-01

Background Heart failure (HF) is a debilitating condition resulting in severe disability and death. In a subset of cases, clustered as Idiopathic Dilated Cardiomyopathy (iDCM), the origin of HF is unknown. In the brain of patients with dementia, proteinaceous aggregates and abnormal oligomeric assemblies of β-amyloid impair cell function and lead to cell death. Methods and Results We have similarly characterized fibrillar and oligomeric assemblies in the hearts of iDCM patients pointing to abnormal protein aggregation as a determinant of iDCM. We also showed that oligomers alter myocyte Ca2+ homeostasis. Additionally, we have identified two new sequence variants in the presenilin-1 (PSEN1) gene promoter leading to reduced gene and protein expression. We also show that presenilin-1 co-immunoprecipitates with SERCA2a. Conclusions Based on these findings we propose that two mechanisms may link protein aggregation and cardiac function: oligomer-induced changes on Ca2+ handling and a direct effect of PSEN1 sequence variants on EC-coupling protein function. PMID:20194882

A universal method for detection of amyloidogenic misfolded proteins.

PubMed

Yam, Alice Y; Wang, Xuemei; Gao, Carol Man; Connolly, Michael D; Zuckermann, Ronald N; Bleu, Thieu; Hall, John; Fedynyshyn, Joseph P; Allauzen, Sophie; Peretz, David; Salisbury, Cleo M

2011-05-24

Diseases associated with the misfolding of endogenous proteins, such as Alzheimer's disease and type II diabetes, are becoming increasingly prevalent. The pathophysiology of these diseases is not totally understood, but mounting evidence suggests that the misfolded protein aggregates themselves may be toxic to cells and serve as key mediators of cell death. As such, an assay that can detect aggregates in a sensitive and selective fashion could provide the basis for early detection of disease, before cellular damage occurs. Here we report the evolution of a reagent that can selectively capture diverse misfolded proteins by interacting with a common supramolecular feature of protein aggregates. By coupling this enrichment tool with protein specific immunoassays, diverse misfolded proteins and sub-femtomole amounts of oligomeric aggregates can be detected in complex biological matrices. We anticipate that this near-universal approach for quantitative misfolded protein detection will become a useful research tool for better understanding amyloidogenic protein pathology as well as serve as the basis for early detection of misfolded protein diseases.

Human High Temperature Requirement Serine Protease A1 (HTRA1) Degrades Tau Protein Aggregates*

PubMed Central

Tennstaedt, Annette; Pöpsel, Simon; Truebestein, Linda; Hauske, Patrick; Brockmann, Anke; Schmidt, Nina; Irle, Inga; Sacca, Barbara; Niemeyer, Christof M.; Brandt, Roland; Ksiezak-Reding, Hanna; Tirniceriu, Anca Laura; Egensperger, Rupert; Baldi, Alfonso; Dehmelt, Leif; Kaiser, Markus; Huber, Robert; Clausen, Tim; Ehrmann, Michael

2012-01-01

Protective proteases are key elements of protein quality control pathways that are up-regulated, for example, under various protein folding stresses. These proteases are employed to prevent the accumulation and aggregation of misfolded proteins that can impose severe damage to cells. The high temperature requirement A (HtrA) family of serine proteases has evolved to perform important aspects of ATP-independent protein quality control. So far, however, no HtrA protease is known that degrades protein aggregates. We show here that human HTRA1 degrades aggregated and fibrillar tau, a protein that is critically involved in various neurological disorders. Neuronal cells and patient brains accumulate less tau, neurofibrillary tangles, and neuritic plaques, respectively, when HTRA1 is expressed at elevated levels. Furthermore, HTRA1 mRNA and HTRA1 activity are up-regulated in response to elevated tau concentrations. These data suggest that HTRA1 is performing regulated proteolysis during protein quality control, the implications of which are discussed. PMID:22535953

Growth hormone aggregates in the rat adenohypophysis

NASA Technical Reports Server (NTRS)

Farrington, M.; Hymer, W. C.

1990-01-01

Although it has been known for some time that GH aggregates are contained within the rat anterior pituitary gland, the role that they might play in pituitary function is unknown. The present study examines this issue using the technique of Western blotting, which permitted visualization of 11 GH variants with apparent mol wt ranging from 14-88K. Electroelution of the higher mol wt variants from gels followed by their chemical reduction with beta-mercaptoethanol increased GH immunoassayability by about 5-fold. With the blot procedure we found 1) that GH aggregates greater than 44K were associated with a 40,000 x g sedimentable fraction; 2) that GH aggregates were not present in glands from thyroidectomized rats, but were in glands from the thyroidectomized rats injected with T4; 3) that GH aggregates were uniquely associated with a heavily granulated somatotroph subpopulation isolated by density gradient centrifugation; and 4) that high mol wt GH forms were released from the dense somatotrophs in culture, since treatment of the culture medium with beta-mercaptoethanol increased GH immunoassayability by about 5-fold. Taken together, the results show that high mol wt GH aggregates are contained in secretory granules of certain somatotrophs and are also released in aggregate form from these cells in vitro.

Heterogeneous Cadherin Expression and Multicellular Aggregate Dynamics in Ovarian Cancer Dissemination.

PubMed

Klymenko, Yuliya; Johnson, Jeffrey; Bos, Brandi; Lombard, Rachel; Campbell, Leigh; Loughran, Elizabeth; Stack, M Sharon

2017-07-01

Epithelial ovarian carcinoma spreads via shedding of cells and multicellular aggregates (MCAs) from the primary tumor into peritoneal cavity, with subsequent intraperitoneal tumor cell:mesothelial cell adhesion as a key early event in metastatic seeding. Evaluation of human tumor extracts and tissues confirms that well-differentiated ovarian tumors express abundant E-cadherin (Ecad), whereas advanced lesions exhibit upregulated N-cadherin (Ncad). Two expression patterns are observed: "mixed cadherin," in which distinct cells within the same tumor express either E- or Ncad, and "hybrid cadherin," wherein single tumor cell(s) simultaneously expresses both cadherins. We demonstrate striking cadherin-dependent differences in cell-cell interactions, MCA formation, and aggregate ultrastructure. Mesenchymal-type Ncad+ cells formed stable, highly cohesive solid spheroids, whereas Ecad+ epithelial-type cells generated loosely adhesive cell clusters covered by uniform microvilli. Generation of "mixed cadherin" MCAs using fluorescently tagged cell populations revealed preferential sorting into cadherin-dependent clusters, whereas mixing of cell lines with common cadherin profiles generated homogeneous aggregates. Recapitulation of the "hybrid cadherin" Ecad+/Ncad+ phenotype, via insertion of the CDH2 gene into Ecad+ cells, resulted in the ability to form heterogeneous clusters with Ncad+ cells, significantly enhanced adhesion to organotypic mesomimetic cultures and peritoneal explants, and increased both migration and matrix invasion. Alternatively, insertion of CDH1 gene into Ncad+ cells greatly reduced cell-to-collagen, cell-to-mesothelium, and cell-to-peritoneum adhesion. Acquisition of the hybrid cadherin phenotype resulted in altered MCA surface morphology with increased surface projections and increased cell proliferation. Overall, these findings support the hypothesis that MCA cadherin composition impacts intraperitoneal cell and MCA dynamics and thereby affects ultimate metastatic success. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Tropomyosin isoform Tpm2.1 regulates collective and amoeboid cell migration and cell aggregation in breast epithelial cells.

PubMed

Shin, HyeRim; Kim, Dayoung; Helfman, David M

2017-11-10

Metastasis dissemination is the result of various processes including cell migration and cell aggregation. These processes involve alterations in the expression and organization of cytoskeletal and adhesion proteins in tumor cells. Alterations in actin filaments and their binding partners are known to be key players in metastasis. Downregulation of specific tropomyosin (Tpm) isoforms is a common characteristic of transformed cells. In this study, we examined the role of Tpm2.1 in non-transformed MCF10A breast epithelial cells in cell migration and cell aggregation, because this isoform is downregulated in primary and metastatic breast cancer as well as various breast cancer cell lines. Downregulation of Tpm2.1 using siRNA or shRNA resulted in retardation of collective cell migration but increase in single cell migration and invasion. Loss of Tpm2.1 is associated with enhanced actomyosin contractility and increased expression of E-cadherin and β-catenin. Furthermore, inhibition of Rho-associated kinase (ROCK) recovered collective cell migration in Tpm2.1-silenced cells. We also found that Tpm2.1-silenced cells formed more compacted spheroids and exhibited faster cell motility when spheroids were re-plated on 2D surfaces coated with fibronectin and collagen. When Tpm2.1 was downregulated, we observed a decrease in the level of AXL receptor tyrosine kinase, which may explain the increased levels of E-cadherin and β-catenin. These studies demonstrate that Tpm2.1 functions as an important regulator of cell migration and cell aggregation in breast epithelial cells. These findings suggest that downregulation of Tpm2.1 may play a critical role during tumor progression by facilitating the metastatic potential of tumor cells.

Kinetics of Lipofuscin Formation in Aging Retinal Pigment Epithelium Cells

NASA Astrophysics Data System (ADS)

Family, Fereydoon; Mazzitello, K. I.; Arizmendi, C. M.; Grossniklaus, Hans E.

2010-03-01

Lipofuscin is a deposit that is formed over time by aggregation and clustering of incompletely degraded membrane material in various types of cells. Lipofuscin is made of free-radical-damaged protein and fat and is known to be present in age- related macular dgeneration (AMD), Alzheimer disease, and Parkinson disease. AMD is the leading cause of blindness in adults. The degradation of retinal pigment epithelium cells (RPE) through accumulation of lipsofuscin is considered a significant pathogenic factor in the development of AMD. We will present the results of a study of the kinetics of lipofuscin growth in RPE cells using Kinetic Monte Carlo simulations and scaling theory on a cluster aggregation model. The model captures the essential physics of lipofuscin growth in the cells. A remarkable feature is that small particles may be removed from the cells while the larger ones become fixed and grow by aggregation. We compare our results with the number of lipofuscin granules in eyes with early age-related degeneration.

A novel Ras-interacting protein required for chemotaxis and cyclic adenosine monophosphate signal relay in Dictyostelium.

PubMed

Lee, S; Parent, C A; Insall, R; Firtel, R A

1999-09-01

We have identified a novel Ras-interacting protein from Dictyostelium, RIP3, whose function is required for both chemotaxis and the synthesis and relay of the cyclic AMP (cAMP) chemoattractant signal. rip3 null cells are unable to aggregate and lack receptor activation of adenylyl cyclase but are able, in response to cAMP, to induce aggregation-stage, postaggregative, and cell-type-specific gene expression in suspension culture. In addition, rip3 null cells are unable to properly polarize in a cAMP gradient and chemotaxis is highly impaired. We demonstrate that cAMP stimulation of guanylyl cyclase, which is required for chemotaxis, is reduced approximately 60% in rip3 null cells. This reduced activation of guanylyl cyclase may account, in part, for the defect in chemotaxis. When cells are pulsed with cAMP for 5 h to mimic the endogenous cAMP oscillations that occur in wild-type strains, the cells will form aggregates, most of which, however, arrest at the mound stage. Unlike the response seen in wild-type strains, the rip3 null cell aggregates that form under these experimental conditions are very small, which is probably due to the rip3 null cell chemotaxis defect. Many of the phenotypes of the rip3 null cell, including the inability to activate adenylyl cyclase in response to cAMP and defects in chemotaxis, are very similar to those of strains carrying a disruption of the gene encoding the putative Ras exchange factor AleA. We demonstrate that aleA null cells also exhibit a defect in cAMP-mediated activation of guanylyl cyclase similar to that of rip3 null cells. A double-knockout mutant (rip3/aleA null cells) exhibits a further reduction in receptor activation of guanylyl cyclase, and these cells display almost no cell polarization or movement in cAMP gradients. As RIP3 preferentially interacts with an activated form of the Dictyostelium Ras protein RasG, which itself is important for cell movement, we propose that RIP3 and AleA are components of a Ras-regulated pathway involved in integrating chemotaxis and signal relay pathways that are essential for aggregation.

Lymphocytes on sounding rocket flights.

PubMed

Cogoli-Greuter, M; Pippia, P; Sciola, L; Cogoli, A

1994-05-01

Cell-cell interactions and the formation of cell aggregates are important events in the mitogen-induced lymphocyte activation. The fact that the formation of cell aggregates is only slightly reduced in microgravity suggests that cells are moving and interacting also in space, but direct evidence was still lacking. Here we report on two experiments carried out on a flight of the sounding rocket MAXUS 1B, launched in November 1992 from the base of Esrange in Sweden. The rocket reached the altitude of 716 km and provided 12.5 min of microgravity conditions.

Cuticular Poroma: A Poroma Mostly Composed of Cuticular Cells (Cuticuloma).

PubMed

Alegría-Landa, Victoria; Kutzner, Heinz; Requena, Luis

2017-12-28

Poromas are benign cutaneous adnexal neoplasms with differentiation toward excretory ducts of eccrine and apocrine glands. They are mainly composed of solid aggregates of small, monomorphous, round, and basophilic poroid cells, with a lower proportion of larger, squamous, eosinophilic cuticular cells, which are lining ductal structures with an eosinophilic luminal cuticle. In most cases of poromas, cuticular cells represent a small proportion of the neoplastic aggregates. We report 2 poromas mostly composed of cuticular cells, and on the basis of these findings, we have named cuticuloma to this histopathologic variant of poroma.

SOD1 oxidation and formation of soluble aggregates in yeast: Relevance to sporadic ALS development

PubMed Central

Martins, Dorival; English, Ann M.

2014-01-01

Misfolding and aggregation of copper–zinc superoxide dismutase (Sod1) are observed in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Mutations in Sod1 lead to familial ALS (FALS), which is a late-onset disease. Since oxidative damage to proteins increases with age, it had been proposed that oxidation of Sod1 mutants may trigger their misfolding and aggregation in FALS. However, over 90% of ALS cases are sporadic (SALS) with no obvious genetic component. We hypothesized that oxidation could also trigger the misfolding and aggregation of wild-type Sod1 and sought to confirm this in a cellular environment. Using quiescent, stationary-phase yeast cells as a model for non-dividing motor neurons, we probed for post-translational modification (PTM) and aggregation of wild-type Sod1 extracted from these cells. By size-exclusion chromatography (SEC), we isolated two populations of Sod1 from yeast: a low-molecular weight (LMW) fraction that is catalytically active and a catalytically inactive, high-molecular weight (HMW) fraction. High-resolution mass spectrometric analysis revealed that LMW Sod1 displays no PTMs but HMW Sod1 is oxidized at Cys146 and His71, two critical residues for the stability and folding of the enzyme. HMW Sod1 is also oxidized at His120, a copper ligand, which will promote loss of this catalytic metal cofactor essential for SOD activity. Monitoring the fluorescence of a Sod1-green-fluorescent-protein fusion (Sod1-GFP) extracted from yeast chromosomally expressing this fusion, we find that HMW Sod1-GFP levels increase up to 40-fold in old cells. Thus, we speculate that increased misfolding and inclusion into soluble aggregates is a consequence of elevated oxidative modifications of wild-type Sod1 as cells age. Our observations argue that oxidative damage to wild-type Sod1 initiates the protein misfolding mechanisms that give rise to SALS. PMID:24936435

Autophagy-independent incorporation of GFP-LC3 into protein aggregates is dependent on its interaction with p62/SQSTM1.

PubMed

Shvets, Elena; Elazar, Zvulun

2008-11-01

LC3 is a widely used marker of autophagosomes in mammalian cells. However, in addition to its autophagosomal localization, GFP-LC3 is often found associated with protein aggregates that are formed in an autophagy-independent manner. In addition, LC3 directly interacts with p62/SQSTM1 (hereafter named p62), a common constituent of protein aggregates. In our recent report, we mapped the regions in LC3 involved in its binding to p62 and showed that this binding is essential for the incorporation of p62 into autophagosomes. Here we demonstrate that the autophagy-unrelated association of GFP-LC3 with protein aggregates is dependent on its interaction with p62.

Heterogeneity within populations of recombinant Chinese hamster ovary cells expressing human interferon-gamma.

PubMed

Coppen, S R; Newsam, R; Bull, A T; Baines, A J

1995-04-20

The Chinese hamster ovary (CHO) cell line has great commercial importance in the production of recombinant human proteins, especially those for therapeutic use. Much attention has been paid to CHO cell population physiology in order to define factors affecting product fidelity and yield. Such studies have revealed that recombinant proteins, including human interferon-gamma (IFN-gamma), can be heterogeneous both in glycosylation and in proteolytic processing. The type of heterogeneity observed depends on the growth physiology of the cell population, although the relationship between them is complex. In this article we report results of a cytological study of the CHO320 line which expresses recombinant human IFN-gamma. When grown in suspension culture, this cell line exhibited three types of heterogeneity: (1) heterogeneity of the production of IFN-gamma within the cell population, (2) heterogeneity of the number of nuclei and mitotic spindles in dividing cells, and (3) heterogeneity of cellular environment. The last of these arises from cell aggregates which form in suspension culture: Some cells are exposed to the culture medium; others are fully enclosed within the mass with little or no direct access to the medium. Thus, live cells producing IFN-gamma are heterogeneous in their environment, with variable access to O(2) and nutrients. Within the aggregates, it appears that live cells proliferate on a dead cell mass. The layer of live cells can be several cells deep. Specific cell-cell attachments are observed between the living cells in these aggregates. Two proteins, known to be required for the formation of certain types of intercellular junctions, spectrin and vinculin, have been localized to the regions of cell-cell contact. The aggregation of the cells appears to be an active process requiring protein synthesis. (c) 1995 John Wiley & Sons, Inc.

Drosophila Melanogaster as a Model System for Studies of Islet Amyloid Polypeptide Aggregation

PubMed Central

Schultz, Sebastian Wolfgang; Nilsson, K. Peter R.; Westermark, Gunilla Torstensdotter

2011-01-01

Background Recent research supports that aggregation of islet amyloid polypeptide (IAPP) leads to cell death and this makes islet amyloid a plausible cause for the reduction of beta cell mass, demonstrated in patients with type 2 diabetes. IAPP is produced by the beta cells as a prohormone, and proIAPP is processed into IAPP by the prohormone convertases PC1/3 and PC2 in the secretory granules. Little is known about the pathogenesis for islet amyloid and which intracellular mechanisms are involved in amyloidogenesis and induction of cell death. Methodology/Principal Findings We have established expression of human proIAPP (hproIAPP), human IAPP (hIAPP) and the non-amyloidogenic mouse IAPP (mIAPP) in Drosophila melanogaster, and compared survival of flies with the expression driven to different cell populations. Only flies expressing hproIAPP in neurons driven by the Gal4 driver elavC155,Gal4 showed a reduction in lifespan whereas neither expression of hIAPP or mIAPP influenced survival. Both hIAPP and hproIAPP expression caused formation of aggregates in CNS and fat body region, and these aggregates were both stained by the dyes Congo red and pFTAA, both known to detect amyloid. Also, the morphology of the highly organized protein granules that developed in the fat body of the head in hIAPP and hproIAPP expressing flies was characterized, and determined to consist of 15.8 nm thick pentagonal rod-like structures. Conclusions/Significance These findings point to a potential for Drosophila melanogaster to serve as a model system for studies of hproIAPP and hIAPP expression with subsequent aggregation and developed pathology. PMID:21695120

Misfolded Polyglutamine, Polyalanine, and Superoxide Dismutase 1 Aggregate via Distinct Pathways in the Cell*

PubMed Central

Polling, Saskia; Mok, Yee-Foong; Ramdzan, Yasmin M.; Turner, Bradley J.; Yerbury, Justin J.; Hill, Andrew F.; Hatters, Danny M.

2014-01-01

Protein aggregation into intracellular inclusions is a key feature of many neurodegenerative disorders. A common theme has emerged that inappropriate self-aggregation of misfolded or mutant polypeptide sequences is detrimental to cell health. Yet protein quality control mechanisms may also deliberately cluster them together into distinct inclusion subtypes, including the insoluble protein deposit (IPOD) and the juxtanuclear quality control (JUNQ). Here we investigated how the intrinsic oligomeric state of three model systems of disease-relevant mutant protein and peptide sequences relates to the IPOD and JUNQ patterns of aggregation using sedimentation velocity analysis. Two of the models (polyalanine (37A) and superoxide dismutase 1 (SOD1) mutants A4V and G85R) accumulated into the same JUNQ-like inclusion whereas the other, polyglutamine (72Q), formed spatially distinct IPOD-like inclusions. Using flow cytometry pulse shape analysis (PulSA) to separate cells with inclusions from those without revealed the SOD1 mutants and 37A to have abruptly altered oligomeric states with respect to the nonaggregating forms, regardless of whether cells had inclusions or not, whereas 72Q was almost exclusively monomeric until inclusions formed. We propose that mutations leading to JUNQ inclusions induce a constitutively “misfolded” state exposing hydrophobic side chains that attract and ultimately overextend protein quality capacity, which leads to aggregation into JUNQ inclusions. Poly(Q) is not misfolded in this same sense due to universal polar side chains, but is highly prone to forming amyloid fibrils that we propose invoke a different engagement mechanism with quality control. PMID:24425868

Misfolded polyglutamine, polyalanine, and superoxide dismutase 1 aggregate via distinct pathways in the cell.

PubMed

Polling, Saskia; Mok, Yee-Foong; Ramdzan, Yasmin M; Turner, Bradley J; Yerbury, Justin J; Hill, Andrew F; Hatters, Danny M

2014-03-07

Protein aggregation into intracellular inclusions is a key feature of many neurodegenerative disorders. A common theme has emerged that inappropriate self-aggregation of misfolded or mutant polypeptide sequences is detrimental to cell health. Yet protein quality control mechanisms may also deliberately cluster them together into distinct inclusion subtypes, including the insoluble protein deposit (IPOD) and the juxtanuclear quality control (JUNQ). Here we investigated how the intrinsic oligomeric state of three model systems of disease-relevant mutant protein and peptide sequences relates to the IPOD and JUNQ patterns of aggregation using sedimentation velocity analysis. Two of the models (polyalanine (37A) and superoxide dismutase 1 (SOD1) mutants A4V and G85R) accumulated into the same JUNQ-like inclusion whereas the other, polyglutamine (72Q), formed spatially distinct IPOD-like inclusions. Using flow cytometry pulse shape analysis (PulSA) to separate cells with inclusions from those without revealed the SOD1 mutants and 37A to have abruptly altered oligomeric states with respect to the nonaggregating forms, regardless of whether cells had inclusions or not, whereas 72Q was almost exclusively monomeric until inclusions formed. We propose that mutations leading to JUNQ inclusions induce a constitutively "misfolded" state exposing hydrophobic side chains that attract and ultimately overextend protein quality capacity, which leads to aggregation into JUNQ inclusions. Poly(Q) is not misfolded in this same sense due to universal polar side chains, but is highly prone to forming amyloid fibrils that we propose invoke a different engagement mechanism with quality control.

GPNMB ameliorates mutant TDP-43-induced motor neuron cell death.

PubMed

Nagahara, Yuki; Shimazawa, Masamitsu; Ohuchi, Kazuki; Ito, Junko; Takahashi, Hitoshi; Tsuruma, Kazuhiro; Kakita, Akiyoshi; Hara, Hideaki

2017-08-01

Glycoprotein nonmetastatic melanoma protein B (GPNMB) aggregates are observed in the spinal cord of amyotrophic lateral sclerosis (ALS) patients, but the detailed localization is still unclear. Mutations of transactive response DNA binding protein 43kDa (TDP-43) are associated with neurodegenerative diseases including ALS. In this study, we evaluated the localization of GPNMB aggregates in the spinal cord of ALS patients and the effect of GPNMB against mutant TDP-43 induced motor neuron cell death. GPNMB aggregates were not localized in the glial fibrillary acidic protein (GFAP)-positive astrocyte and ionized calcium binding adaptor molecule-1 (Iba1)-positive microglia. GPNMB aggregates were localized in the microtubule-associated protein 2 (MAP-2)-positive neuron and neurofilament H non-phosphorylated (SMI-32)-positive neuron, and these were co-localized with TDP-43 aggregates in the spinal cord of ALS patients. Mock or TDP-43 (WT, M337V, and A315T) plasmids were transfected into mouse motor neuron cells (NSC34). The expression level of GPNMB was increased by transfection of mutant TDP-43 plasmids. Recombinant GPNMB ameliorated motor neuron cell death induced by transfection of mutant TDP-43 plasmids and serum-free stress. Furthermore, the expression of phosphorylated ERK1/2 and phosphorylated Akt were decreased by this stress, and these expressions were increased by recombinant GPNMB. These results indicate that GPNMB has protective effects against mutant TDP-43 stress via activating the ERK1/2 and Akt pathways, and GPNMB may be a therapeutic target for TDP-43 proteinopathy in familial and sporadic ALS. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Hsp105 reduces the protein aggregation and cytotoxicity by expanded-polyglutamine proteins through the induction of Hsp70

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

Yamagishi, Nobuyuki; Goto, Kazumasa; Nakagawa, Satomi

2010-09-10

Hsp105{alpha} and Hsp105{beta} are major heat shock proteins in mammalian cells and belong to the HSP105/110 family. Hsp105{alpha} is expressed constitutively in the cytoplasm of cells, while Hsp105{beta}, an alternatively spliced form of Hsp105{alpha}, is expressed specifically in the nucleus of cells during mild heat shock. Here, we show that not only Hsp105{beta} but also Hsp105{alpha} accumulated in the nucleus of cells following the expression of enhanced green fluorescent protein with a pathological length polyQ tract (EGFP-polyQ97) and suppressed the intranuclear aggregation of polyQ proteins and apoptosis induced by EGFP-polyQ97. Mutants of Hsp105{alpha} and Hsp105{beta} with changes in the nuclearmore » localization signal sequences, which localized exclusively in the cytoplasm with or without the expression of EGFP-polyQ97, did not suppress the intranuclear aggregation of polyQ proteins and apoptosis induced by EGFP-polyQ97. Furthermore, Hsp70 was induced by the co-expression of Hsp105{alpha} and EGFP-polyQ97, and the knockdown of Hsp70 reduced the inhibitory effect of Hsp105{alpha} and Hsp105{beta} on the intranuclear aggregation of polyQ proteins and apoptosis induced by EGFP-polyQ97. These observations suggested that Hsp105{alpha} and Hsp105{beta} suppressed the expanded polyQ tract-induced protein aggregation and apoptosis through the induction of Hsp70.« less

Analysis of the cellulose synthase operon genes, bcsA, bcsB, and bcsC in Cronobacter species: Prevalence among species and their roles in biofilm formation and cell-cell aggregation.

PubMed

Hu, Lan; Grim, Christopher J; Franco, Augusto A; Jarvis, Karen G; Sathyamoorthy, Vengopal; Kothary, Mahendra H; McCardell, Barbara A; Tall, Ben D

2015-12-01

Cronobacter species are emerging food-borne pathogens that cause severe sepsis, meningitis, and necrotizing entercolitis in neonates and infants. Bacterial pathogens such as Escherichia coli and Salmonella species produce extracellular cellulose which has been shown to be involved in rugosity, biofilm formation, and host colonization. In this study the distribution and prevalence of cellulose synthase operon genes (bcsABZC) were determined by polymerase chain reaction (PCR) analysis in 231 Cronobacter strains isolated from clinical, food, environmental, and unknown sources. Furthermore, bcsA and bcsB isogenic mutants were constructed in Cronobacter sakazakii BAA894 to determine their roles. In calcofluor binding assays bcsA and bcsB mutants did not produce cellulose, and their colonial morphotypes were different to that of the parent strain. Biofilm formation and bacterial cell-cell aggregation were significantly reduced in bcsA and bcsB mutants compared to the parental strain. bcsA or bcsAB PCR-negative strains of C. sakazakii did not bind calcofluor, and produced less biofilm and cell-cell aggregation compared to strains possessing bcsAB genes. These data indicated that Cronobacter bcsABZC were present in all clinical isolates and most of food and environmental isolates. bcsA and bcsB genes of Cronobacter were necessary to produce cellulose, and were involved in biofilm formation and cell-cell aggregation. Published by Elsevier Ltd.

The spread of prion-like proteins by lysosomes and tunneling nanotubes: Implications for neurodegenerative diseases.

PubMed

Victoria, Guiliana Soraya; Zurzolo, Chiara

2017-09-04

Progression of pathology in neurodegenerative diseases is hypothesized to be a non-cell-autonomous process that may be mediated by the productive spreading of prion-like protein aggregates from a "donor cell" that is the source of misfolded aggregates to an "acceptor cell" in which misfolding is propagated by conversion of the normal protein. Although the proteins involved in the various diseases are unrelated, common pathways appear to be used for their intercellular propagation and spreading. Here, we summarize recent evidence of the molecular mechanisms relevant for the intercellular trafficking of protein aggregates involved in prion, Alzheimer's, Huntington's, and Parkinson's diseases. We focus in particular on the common roles that lysosomes and tunneling nanotubes play in the formation and spreading of prion-like assemblies. © 2017 Victoria and Zurzolo.

Replacement of charged and polar residues in the coiled-coiled interface of huntingtin-interacting protein 1 (HIP1) causes aggregation and cell death.

PubMed

Fontaine, Sarah N; Bauer, Scott P; Lin, Xiaoyan; Poorfarahani, Sara; Ybe, Joel A

2012-09-21

HIP1 crystal structures solved in our laboratory revealed abnormalities in the coiled-coil region, suggesting intrinsic plasticity. To test this, specific amino acids in the coiled-coil were mutated. The apparent thermal stability of HIP1 was altered when Thr528 and Glu531 were replaced by leucine, and was enhanced when Lys510 was also mutated. In cells, HIP1 mutant expression produced aggregation. MTS and flow cytometry indicate a correlation between aggregated HIP1 and enhanced cell death. These data support the idea that flexibility of the HIP1 coiled-coil domain is important for normal function and may lead to new insights into Huntington's disease. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Cell fiber-based three-dimensional culture system for highly efficient expansion of human induced pluripotent stem cells.

PubMed

Ikeda, Kazuhiro; Nagata, Shogo; Okitsu, Teru; Takeuchi, Shoji

2017-06-06

Human pluripotent stem cells are a potentially powerful cellular resource for application in regenerative medicine. Because such applications require large numbers of human pluripotent stem cell-derived cells, a scalable culture system of human pluripotent stem cell needs to be developed. Several suspension culture systems for human pluripotent stem cell expansion exist; however, it is difficult to control the thickness of cell aggregations in these systems, leading to increased cell death likely caused by limited diffusion of gases and nutrients into the aggregations. Here, we describe a scalable culture system using the cell fiber technology for the expansion of human induced pluripotent stem (iPS) cells. The cells were encapsulated and cultured within the core region of core-shell hydrogel microfibers, resulting in the formation of rod-shaped or fiber-shaped cell aggregations with sustained thickness and high viability. By encapsulating the cells with type I collagen, we demonstrated a long-term culture of the cells by serial passaging at a high expansion rate (14-fold in four days) while retaining its pluripotency. Therefore, our culture system could be used for large-scale expansion of human pluripotent stem cells for use in regenerative medicine.

GD1a Overcomes Inhibition of Myelination by Fibronectin via Activation of Protein Kinase A: Implications for Multiple Sclerosis.

PubMed

Qin, Jing; Sikkema, Arend H; van der Bij, Kristine; de Jonge, Jenny C; Klappe, Karin; Nies, Vera; Jonker, Johan W; Kok, Jan Willem; Hoekstra, Dick; Baron, Wia

2017-10-11

Remyelination failure by oligodendrocytes contributes to the functional impairment that characterizes the demyelinating disease multiple sclerosis (MS). Since incomplete remyelination will irreversibly damage axonal connections, treatments effectively promoting remyelination are pivotal in halting disease progression. Our previous findings suggest that fibronectin aggregates, as an environmental factor, contribute to remyelination failure by perturbing oligodendrocyte progenitor cell (OPC) maturation. Here, we aim at elucidating whether exogenously added gangliosides (i.e., cell surface lipids with a potential to modulate signaling pathways) could counteract fibronectin-mediated inhibition of OPC maturation. Exclusive exposure of rat oligodendrocytes to GD1a, but not other gangliosides, overcomes aggregated fibronectin-induced inhibition of myelin membrane formation, in vitro , and OPC differentiation in fibronectin aggregate containing cuprizone-induced demyelinated lesions in male mice. GD1a exerts its effect on OPCs by inducing their proliferation and, at a late stage, by modulating OPC maturation. Kinase activity profiling revealed that GD1a activated a protein kinase A (PKA)-dependent signaling pathway and increased phosphorylation of the transcription factor cAMP response element-binding protein. Consistently, the effect of GD1a in restoring myelin membrane formation in the presence of fibronectin aggregates was abolished by the PKA inhibitor H89, whereas the effect of GD1a was mimicked by the PKA activator dibutyryl-cAMP. Together, GD1a overcomes the inhibiting effect of aggregated fibronectin on OPC maturation by activating a PKA-dependent signaling pathway. Given the persistent presence of fibronectin aggregates in MS lesions, ganglioside GD1a might act as a potential novel therapeutic tool to selectively modulate the detrimental signaling environment that precludes remyelination. SIGNIFICANCE STATEMENT As an environmental factor, aggregates of the extracellular matrix protein fibronectin perturb the maturation of oligodendrocyte progenitor cells (OPCs), thereby impeding remyelination, in the demyelinating disease multiple sclerosis (MS). Here we demonstrate that exogenous addition of ganglioside GD1a overcomes the inhibiting effect of aggregated fibronectin on OPC maturation, both in vitro and in vivo , by activating a PKA-dependent signaling pathway. We propose that targeted delivery of GD1a to MS lesions may act as a potential novel molecular tool to boost maturation of resident OPCs to overcome remyelination failure and halt disease progression. Copyright © 2017 the authors 0270-6474/17/379925-14$15.00/0.

Heterologous Gln/Asn-Rich Proteins Impede the Propagation of Yeast Prions by Altering Chaperone Availability

PubMed Central

Yang, Zi; Hong, Joo Y.; Derkatch, Irina L.; Liebman, Susan W.

2013-01-01

Prions are self-propagating conformations of proteins that can cause heritable phenotypic traits. Most yeast prions contain glutamine (Q)/asparagine (N)-rich domains that facilitate the accumulation of the protein into amyloid-like aggregates. Efficient transmission of these infectious aggregates to daughter cells requires that chaperones, including Hsp104 and Sis1, continually sever the aggregates into smaller “seeds.” We previously identified 11 proteins with Q/N-rich domains that, when overproduced, facilitate the de novo aggregation of the Sup35 protein into the [PSI +] prion state. Here, we show that overexpression of many of the same 11 Q/N-rich proteins can also destabilize pre-existing [PSI +] or [URE3] prions. We explore in detail the events leading to the loss (curing) of [PSI+] by the overexpression of one of these proteins, the Q/N-rich domain of Pin4, which causes Sup35 aggregates to increase in size and decrease in transmissibility to daughter cells. We show that the Pin4 Q/N-rich domain sequesters Hsp104 and Sis1 chaperones away from the diffuse cytoplasmic pool. Thus, a mechanism by which heterologous Q/N-rich proteins impair prion propagation appears to be the loss of cytoplasmic Hsp104 and Sis1 available to sever [PSI +]. PMID:23358669

Engineered antibody CH2 domains binding to nucleolin: Isolation, characterization and improvement of aggregation.

PubMed

Li, Dezhi; Gong, Rui; Zheng, Jun; Chen, Xihai; Dimitrov, Dimiter S; Zhao, Qi

2017-04-01

Smaller recombinant antibody fragments are now emerging as alternatives of conventional antibodies. Especially, immunoglobulin (Ig) constant CH2 domain and engineered CH2 with improved stability are promising as scaffolds for selection of specific binders to various antigens. We constructed a yeast display library based on an engineered human IgG1 CH2 scaffold with diversified loop regions. A group of CH2 binders were isolated from this yeast display library by panning against nucleolin, which is a tumor-associated antigen involved in cell proliferation, tumor cell growth and angiogenesis. Out of 20 mutants, we selected 3 clones exhibiting relatively high affinities to nucleolin on yeasts. However, recombinant CH2 mutants aggregated when they were expressed. To find the mechanism of the aggregation, we employed computational prediction approaches through structural homology models of CH2 binders. The analysis of potential aggregation prone regions (APRs) and solvent accessible surface areas (ASAs) indicated two hydrophobic residues, Val 264 and Leu 309 , in the β-sheet, in which replacement of both charged residues led to significant decrease of the protein aggregation. The newly identified CH2 binders could be improved to use as candidate therapeutics or research reagents in the future. Copyright © 2017 Elsevier Inc. All rights reserved.

Regulation of microtubule-based transport by MAP4

PubMed Central

Semenova, Irina; Ikeda, Kazuho; Resaul, Karim; Kraikivski, Pavel; Aguiar, Mike; Gygi, Steven; Zaliapin, Ilya; Cowan, Ann; Rodionov, Vladimir

2014-01-01

Microtubule (MT)-based transport of organelles driven by the opposing MT motors kinesins and dynein is tightly regulated in cells, but the underlying molecular mechanisms remain largely unknown. Here we tested the regulation of MT transport by the ubiquitous protein MAP4 using Xenopus melanophores as an experimental system. In these cells, pigment granules (melanosomes) move along MTs to the cell center (aggregation) or to the periphery (dispersion) by means of cytoplasmic dynein and kinesin-2, respectively. We found that aggregation signals induced phosphorylation of threonine residues in the MT-binding domain of the Xenopus MAP4 (XMAP4), thus decreasing binding of this protein to MTs. Overexpression of XMAP4 inhibited pigment aggregation by shortening dynein-dependent MT runs of melanosomes, whereas removal of XMAP4 from MTs reduced the length of kinesin-2–dependent runs and suppressed pigment dispersion. We hypothesize that binding of XMAP4 to MTs negatively regulates dynein-dependent movement of melanosomes and positively regulates kinesin-2–based movement. Phosphorylation during pigment aggregation reduces binding of XMAP4 to MTs, thus increasing dynein-dependent and decreasing kinesin-2–dependent motility of melanosomes, which stimulates their accumulation in the cell center, whereas dephosphorylation of XMAP4 during dispersion has an opposite effect. PMID:25143402

Epigallocatechin-3-gallate (EGCG)-stabilized selenium nanoparticles coated with Tet-1 peptide to reduce amyloid-β aggregation and cytotoxicity.

PubMed

Zhang, Jingnan; Zhou, Xianbo; Yu, Qianqian; Yang, Licong; Sun, Dongdong; Zhou, Yanhui; Liu, Jie

2014-06-11

Alzheimer's disease (AD), the most common neurodegenerative disease, is caused by an accumulation of amyloid-β (Aβ) plaque deposits in the brains. Evidence is increasingly showing that epigallocatechin-3-gallate (EGCG) can partly protect cells from Aβ-mediated neurotoxicity by inhibiting Aβ aggregation. In order to better understand the process of Aβ aggregation and amyloid fibril disaggregation and reduce the cytotoxicity of EGCG at high doses, we attached EGCG onto the surface of selenium nanoparticles (EGCG@Se). Given the low delivery efficiency of EGCG@Se to the targeted cells and the involvement of selenoprotein in antioxidation and neuroprotection, which are the key factors for preventing the onset and progression of AD, we synthesized EGCG-stabilized selenium nanoparticles coated with Tet-1 peptide (Tet-1-EGCG@Se, a synthetic selenoprotein analogue), considering the affinity of Tet-1 peptide to neurons. We revealed that Tet-1-EGCG@Se can effectively inhibit Aβ fibrillation and disaggregate preformed Aβ fibrils into nontoxic aggregates. In addition, we found that both EGCG@Se and Tet-1-EGCG@Se can label Aβ fibrils with a high affinity, and Tet-1 peptides can significantly enhance the cellular uptake of Tet-1-EGCG@Se in PC12 cells rather than in NIH/3T3 cells.

Hsp25, a member of the Hsp30 family, promotes inclusion formation in response to stress.

PubMed

Katoh, Yumiko; Fujimoto, Mitsuaki; Nakamura, Kosuke; Inouye, Sachiye; Sugahara, Kazuma; Izu, Hanae; Nakai, Akira

2004-05-07

Protein aggregates are oligomeric complexes of misfolded proteins, and serve as the seeds of inclusion bodies termed aggresomes in the cells. Heat shock proteins (Hsps) prevent misfolding and aggregate formation. Here, we found that only avian Hsp25 dominantly accumulated in the aggresomes induced by proteasome inhibition. Molecular cloning of chicken Hsp25 (cHsp25) revealed that it belongs to the Hsp30 family, which is a subfamily of the alpha-crystallin/small Hsp gene family. Unexpectedly, overexpression of cHsp25 into HeLa cells promoted inclusion formation whereas overexpression of mouse Hsp27 and its chicken homologue did not. These results suggest that cHsp25 acts differently from other small Hsps on protein aggregates.

Studies on bacterial inclusion bodies.

PubMed

de Groot, Natalia S; Espargaró, Alba; Morell, Montserrat; Ventura, Salvador

2008-08-01

The field of protein misfolding and aggregation has become an extremely active area of research in recent years. Of particular interest is the deposition of polypeptides into inclusion bodies inside bacterial cells. One reason for this interest is that protein aggregation constitutes a major bottleneck in protein production and restricts the spectrum of protein-based drugs available for commercialization. Additionally, prokaryotic cells could provide a simple yet powerful system for studying the formation and prevention of toxic aggregates, such as those responsible for a number of degenerative diseases. Here, we review recent work that has challenged our understanding of the structure and physiology of inclusion bodies and provided us with a new view of intracellular protein deposition, which has important implications in microbiology, biomedicine and biotechnology.

Mobility Enhancement of Red Blood Cells with Biopolymers

NASA Astrophysics Data System (ADS)

Tahara, Daiki; Oikawa, Noriko; Kurita, Rei

2016-03-01

Adhesion of red blood cells (RBC) to substrates are one of crucial problems for a blood clot. Here we investigate the mobility of RBC between two glass substrates in saline with polymer systems. We find that RBCs are adhered to the glass substrate with PEG, however the mobility steeply increases with fibrinogen and dextran, which are biopolymers. We also find that the mobility affects an aggregation dynamics of RBCs, which is related with diseases such as influenza, blood clot and so on. The Brownian motion helps to increase probability of contact with each other and to find a more stable condition of the aggregation. Thus the biopolymers play important roles not only for preventing the adhesion but also for the aggregation.