Role and function of nonmuscle alpha-actinin-1 and -4 in regulating distinct subcategories of actin stress fibers in mammalian cells

Actin stress fibers are dynamic structures in the cytoskeleton, which respond to mechanical stimuli and affect cell motility, adhesion and invasion of cancer cells. In nonmuscle cells, stress fibers have been subcategorized to three distinct stress fiber types: dorsal and ventral stress fibers and transverse arcs. These stress fibers are dissimilar in their subcellular localization, connection to substratum as well as in their dynamics and assembly mechanisms. Still uncharacterized is how they differ in their function and molecular composition. Here, I have studied involvement of nonmuscle alpha-actinin-1 and -4 in regulating distinct stress fibers as well as their localization and function in human U2OS osteosarcoma cells. Except for the correlation of upregulation of alpha-actinin-4 in invasive cancer types very little is known about whether these two actinins are redundant or have specific roles. The availability of highly specific alpha-actinin-1 antibody generated in the lab, revealed localization of alpha-actinin-1 along all three categories of stress fibers while alphaactinin-4 was detected at cell edge, distal ends of stress fibers as well as perinuclear regions. Strikingly, by utilizing RNAi-mediated gene silencing of alpha-actinin-1 resulted in specific loss of dorsal stress fibers and relocalization of alpha-actinin-4 to remaining transverse arcs and ventral stress fibers. Unexpectedly, aberrant migration was not detected in cells lacking alpha-actinin-1 even though focal adhesions were significantly smaller and fewer. Whereas, silencing of alpha-actinin-4 noticeably affected overall cell migration. In summary, as part of my master thesis study I have been able to demonstrate distinct localization and functional patterns for both alpha-actinin-1 and -4. I have identified alpha-actinin-1 to be a selective dorsal stress fiber crosslinking protein as well as to be required for focal adhesion maturation, while alpha-actinin-4 was demonstrated to be fundamental for cell migration.

Dielectric relaxation and electrical conductivity in Bi5NbO10 oxygen ion conductors prepared by a modified sol–gel process

Crystalline Bi5NbO10 nanoparticles have been achieved through a modified sol–gel process using a mixture of ethylenediamine and ethanolamine as a solvent. The Bi5NbO10 nanoparticles were characterized by X-ray diffraction (XRD), differential scanning calorimetry/thermogravimetry (DSC/TG), Fourier transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM) and Raman spectroscopy. The results showed that well-dispersed 5–60 nm Bi5NbO10 nanoparticles were prepared through heat-treating the precursor at 650 °C and the high density pellets were obtained at temperatures lower than those commonly employed. The frequency and temperature dependence of the dielectric constant and the electrical conductivity of the Bi5NbO10 solid solutions were investigated in the 0.1 Hz to 1 MHz frequency range. Two distinct relaxation mechanisms were observed in the plots of dielectric loss and the imaginary part of impedance (Z″) versus frequency in the temperature range of 200–350 °C. The dielectric constant and the loss in the low frequency regime were electrode dependent. The ionic conductivity of Bi5NbO10 solid solutions at 700 °C is 2.86 Ω−1 m−1 which is in same order of magnitude for Y2O3-stabilized ZrO2 ceramics at same temperature. These results suggest that Bi5NbO10 is a promising material for an oxygen ion conductor.

Normal vibrations of N,N-dimethylacetamide

Infrared and Raman spectra of N,N-dimethylacetamide (DMA) are recorded and the normal vibrational analysis of the DMA skeleton as well as the entire molecule carried out employing the Urey-Bradley and modified Urey-Bradley force fields. Vibrational frequencies are assigned on the basis of the normal coordinate calculations and are compared with those of related molecules. Infrared spectra of metal complexes are examined to substantiate the band assignments.

Aspects of tautomerism. Part 16. Influence of the y-keto function on the reactions of sulphonic acids

Reaction of sodium 2-formylbenzenesulphonate (1) with thionyl chloride or phosphorous pentachloride gives a mixture of pseudo (2) and normal (3) sulphonyl chlorides. Whereas ammonium 2-carboxybenzenesulphonate (6) gives only the normal sulphonyl chloride (7) on reaction with thionyl chloride, a mixture of normal (7) and pseudo (8) isomers are formed on reaction with phosphorous pentachloride. Sodium 2-benzoylbenzenesulphonate (15), on the other hand, gives the corresponding normal sulphonyl chloride (16) on reaction with both of the reagents mentioned above. Based on these observations it is concluded that γ-keto sulphonic acids are amenable to the influence of γ-carbonyl group as in the case of γ-keto carboxylic acids but to a lesser extent. © 1989 Indian Academy of Sciences.

Systematic bias in MODIS dust aerosol retrieval at Kanpur (AERONET), Indo-Gangetic Basin - art.no.640816

We have compared the spectral aerosol optical depth (AOD) and aerosol fine mode fraction (AFMF) derived from Moderate Resolution Imaging Spectroradiometer (MODIS) with those of Aerosol Robotic Network (AERONET) at Kanpur (26.45N, 80.35E), northern India for the pre-monsoon season (March to June, 2001-2005). We found that MODIS systematically overestimates AOD during pre-monsoon season (known to be influenced by dust transport from north-west of India). The errors in AOD were correlated with the MODIS top-of-atmosphere apparent surface reflectance in 2.1 mu m channel (rho*(2.1)). MODIS aerosol algorithm uses p*(2.1) to derive the surface reflectance in visible channels (rho(0.47), rho(0.66)) using an empirical mid IR-visible correlation (rho(0.47) = rho(2.1)/4, rho(0.66) = rho(2.1)/2). The large uncertainty in estimating surface reflectance in visible channels (Delta rho(0.66)+/- 0.04, Delta rho(0.47)+/- 0.02) at higher values of p*(2.1) (p*(2.1) > 0.18) leads to higher aerosol contribution in the total reflected radiance at top-of atmosphere to compensate for the reduced surface reflectance in visible channels and thus leads to overestimation of AOD. This was also reflected in the very low values of AFMF during pre-monsoon whose accuracy depends on the aerosol path radiance in 0.47 and 0.66 mu m channels and aerosol models. The errors in AOD were also high in the scattering angle range 110 degrees-140 degrees, where the effect of dust non-spherity on its optical properties is significant. The direct measurements of spectral surface reflectance are required over the Indo-Gangetic basin in order to validate the mid IR-visible relationship. MODIS aerosol models should also be modified to incorporate the effect of non-spherity of dust aerosols.

Analysis of a thick plate with a circular hole resting on a smooth rigid bed and subjected to axisymmetric normal load

A solution for the stresses and displacements in an radially infinite thick plate having a circular hole, one face of which resting on a smooth rigid bed and the other face subjected to axisymmetric normal loading is given. The solution is obtained in terms of Fourier-Bessel series and integral for the Love's stress function. Numerical results are presented for one particular ratio of thickness of plate to the hole radius and loading. It is also shown that the Poisson's ratio has a predominant effect on certain stresses and displacements. The solution would be useful in the stress analysis of bolted joints.Eine Lösung für die Spannungen und Verschiebungen in einer radial, unendlich ausgedehnten, dicken Platte mit einem kreisförmigen Loch, wobei eine Seite auf einer ebenen, starren Unterlage aufliegt, die andere Seite durch eine achsensymmetrische Vertikallast belastet ist, wird angegeben. Die Lösung wird in Form von Fourier-Bessel-Reihen und Integralen der Loveschen Spannungsfunktion angegeben. Numerische Ergebnisse werden für ein bestimmtes Verhältnis der Plattendicke zum Lochradius sowie zur Belastung angegeben. Es wird auch gezeigt, daß das Poisssonsche Verhältnis einen besonderen Einfluß auf bestimmte Spannungen und Verschiebungen hat. Die Lösung ist anwendbar für die Spannungsermittlung von Bolzenverbindungen.

Medium access control schemes for local area networks with multiple priority function

This paper deals with the development and performance evaluation of three modified versions of a scheme proposed for medium access control in local area networks. The original scheme implements a collision-free and fair medium arbitration by using a control wire in conjunction with a data bus. The modifications suggested in this paper are intended to realize the multiple priority function in local area networks.

First-principles analysis of electron correlation, spin ordering and phonons in the normal state of FeSe1-x

We present first-principles density-functional-theory-based calculations to determine the effects of the strength of on-site electron correlation, magnetic ordering, pressure and Se vacancies on phonon frequencies and electronic structure of FeSe1-x. The theoretical equilibrium structure (lattice parameters) of FeSe depends sensitively on the value of the Hubbard parameter U of on-site correlation and magnetic ordering. Our results suggest that there is a competition between different antiferromagnetic states due to comparable magnetic exchange couplings between first- and second-neighbor Fe sites. As a result, a short range order of stripe antiferromagnetic type is shown to be relevant to the normal state of FeSe at low temperature. We show that there is a strong spin-phonon coupling in FeSe (comparable to its superconducting transition temperature) as reflected in large changes in the frequencies of certain phonons with different magnetic ordering, which is used to explain the observed hardening of a Raman-active phonon at temperatures (similar to 100 K) where magnetic ordering sets in. The symmetry of the stripe antiferromagnetic phase permits an induced stress with orthorhombic symmetry, leading to orthorhombic strain as a secondary order parameter at the temperature of magnetic ordering. The presence of Se vacancies in FeSe gives rise to a large peak in the density of states near the Fermi energy, which could enhance the superconducting transition temperature within the BCS-like picture.

Dynamics of sheared inelastic dumbbells

We study the dynamical properties of the homogeneous shear flow of inelastic dumbbells in two dimensions as a first step towards examining the effect of shape on the properties of flowing granular materials. The dumbbells are modelled as smooth fused disks characterized by the ratio of the distance between centres (L) and the disk diameter (D), with an aspect ratio (L/D) varying between 0 and 1 in our simulations. Area fractions studied are in the range 0.1-0.7, while coefficients of normal restitution (e(n)) from 0.99 to 0.7 are considered. The simulations use a modified form of the event-driven methodology for circular disks. The average orientation is characterized by an order parameter S, which varies between 0 (for a perfectly disordered fluid) and 1 (for a fluid with the axes of all dumbbells in the same direction). We investigate power-law fits of S as a function of (L D) and (1 - e(n)(2)) There is a gradual increase in ordering as the area fraction is increased, as the aspect ratio is increased or as the coefficient of restitution is decreased. The order parameter has a maximum value of about 0.5 for the highest area fraction and lowest coefficient of restitution considered here. The mean energy of the velocity fluctuations in the flow direction is higher than that in the gradient direction and the rotational energy, though the difference decreases as the area fraction increases, due to the efficient collisional transfer of energy between the three directions. The distributions of the translational and rotational velocities are Gaussian to a very good approximation. The pressure is found to be remarkably independent of the coefficient of restitution. The pressure and dissipation rate show relatively little variation when scaled by the collision frequency for all the area fractions studied here, indicating that the collision frequency determines the momentum transport and energy dissipation, even at the lowest area fractions studied here. The mean angular velocity of the particles is equal to half the vorticity at low area fractions, but the magnitude systematically decreases to less than half the vorticity as the area fraction is increased, even though the stress tensor is symmetric.

Regulation and Function of GATA Transcription Factors in Adrenocortical Tumors and Granulosa Cells

Transcription factors play a key role in tumor development, in which dysfunction of genes regulating tissue growth and differentiation is a central phenomenon. The GATA family of transcription factors consists of six members that bind to a consensus DNA sequence (A/T)GATA(A/G) in gene promoters and enhancers. The two GATA factors expressed in the adrenal cortex are GATA-4 and GATA-6. In both mice and humans, GATA-4 can be detected only during the fetal period, whereas GATA-6 expression is abundant both throughout development and in the adult. It is already established that GATA factors are important in both normal development and tumorigenesis of several endocrine organs, and expression of GATA-4 and GATA-6 is detected in adrenocortical tumors. The aim of this study was to elucidate the function of these factors in adrenocortical tumor growth. In embryonal development, the adrenocortical cells arise and differentiate from a common pool with gonadal steroidogenic cells, the urogenital ridge. As the adult adrenal cortex undergoes constant renewal, it is hypothesized that undifferentiated adrenocortical progenitor cells reside adjacent to the adrenal capsule and give rise to daughter cells that differentiate and migrate centripetally. A diverse array of hormones controls the differentiation, growth and survival of steroidogenic cells in the adrenal gland and the gonads. Factors such as luteinizing hormone and inhibins, traditionally associated with gonadal steroidogenic cells, can also influence the function of adrenocortical cells in physiological and pathophysiological states. Certain inbred strains of mice develop subcapsular adrenocortical tumors in response to gonadectomy. In this study, we found that these tumors express GATA-4, normally absent from the adult adrenal cortex, while GATA-6 expression is downregulated. Gonadal markers such as luteinizing hormone receptor, anti-Müllerian hormone and P450c17 are also expressed in the neoplastic cells, and the tumors produce gonadal hormones. The tumor cells have lost the expression of melanocortin-2 receptor and the CYP enzymes necessary for the synthesis of corticosterone and aldosterone. By way of xenograft studies utilizing NU/J nude mice, we confirmed that chronic gonadotropin elevation is sufficient to induce adrenocortical tumorigenesis in susceptible inbred strains. Collectively, these studies suggest that subcapsular adrenocortical progenitor cells can, under certain conditions, adopt a gonadal fate. We studied the molecular mechanisms involved in gene regulation in endocrine cells in order to elucidate the role of GATA factors in endocrine tissues. Ovarian granulosa cells express both GATA-4 and GATA-6, and the TGF-β signaling pathway is active in these cells. Inhibin-α is both a target gene for, and an atypical or antagonistic member of the TGF-β growth factor superfamily. In this study, we show that GATA-4 is required for TGF-β-mediated inhibin-α promoter activation in granulosa cells, and that GATA-4 physically interacts with Smad3, a TGF-β downstream protein. Apart from the regulation of steroidogenesis and other events in normal tissues, TGF-β signaling is implicated in tumors of multiple organs, including the adrenal cortex. Another signaling pathway found often to be aberrantly active in adrenocortical tumors is the Wnt pathway. As both of these pathways regulate the expression of inhibin-α, a transcriptional target for GATA-4 and GATA-6, we wanted to investigate whether GATA factors are associated with the components of these signaling cascades in human adrenocortical tumors. We found that the expression of Wnt co-receptors LRP5 and LRP6, Smad3, GATA-6 and SF-1 was diminished in adrenocortical carcinomas with poor outcome. All of these factors drive inhibin-α expression, and their expression in adrenocortical tumors correlated with that of inhibin-α. The results support a tumor suppressor role previously suggested for inhibin-α in the mouse adrenal cortex, and offer putative pathways associated with adrenocortical tumor aggressiveness. Unraveling the role of GATA factors and associated molecules in human and mouse adrenocortical tumors could ultimately contribute to the development of diagnostic tools and future therapies for these diseases.

Exact path-integral evaluation of the heat distribution function of a trapped Brownian oscillator

Using path integrals, we derive an exact expression-valid at all times t-for the distribution P(Q,t) of the heat fluctuations Q of a Brownian particle trapped in a stationary harmonic well. We find that P(Q, t) can be expressed in terms of a modified Bessel function of zeroth order that in the limit t > infinity exactly recovers the heat distribution function obtained recently by Imparato et al. Phys. Rev. E 76, 050101(R) (2007)] from the approximate solution to a Fokker-Planck equation. This long-time result is in very good agreement with experimental measurements carried out by the same group on the heat effects produced by single micron-sized polystyrene beads in a stationary optical trap. An earlier exact calculation of the heat distribution function of a trapped particle moving at a constant speed v was carried out by van Zon and Cohen Phys. Rev. E 69, 056121 (2004)]; however, this calculation does not provide an expression for P(Q, t) itself, but only its Fourier transform (which cannot be analytically inverted), nor can it be used to obtain P(Q, t) for the case v=0.

Autoimmune Regulator (AIRE) in the Maintenance of Immunological Tolerance

Autoimmune regulator (AIRE) is the gene mutated in the human polyglandular autoimmune disease called Autoimmune polyendocrinopathy, candidiasis, and ectodermal dystrophy (APECED) that belongs to the Finnish disease heritage. Murine Aire has been shown to be important in the generation of the T cell central tolerance in the thymus by promoting the expression of ectopic tissue-specific antigens in the thymic medulla. Aire is also involved in the thymus tissue organization during organogenesis. In addition to the thymus, AIRE/Aire is expressed in the secondary lymphoid organs. Accordingly, a role for AIRE/Aire in the maintenance of peripheral tolerance has been suggested. Peripheral tolerance involves mechanisms that suppress immune responses in secondary lymphoid organs. Regulatory T cells (Tregs) are an important suppressive T cell population mediating the peripheral tolerance. Tregs are generated in the thymus but also in the peripheral immune system T cells can acquire the Treg-phenotype. The aim of this study was to characterize Tregs in APECED patients and in the APECED mouse model (Aire-deficient mice). In the mouse model, it was possible to separate Aire expression in the thymus and in the secondary lymphoid organs. The relative importance of thymic and peripheral Aire expression in the maintenance of immunological tolerance was studied in an experimental model that was strongly biased towards autoimmunity, i.e. lymphopenia-induced proliferation (LIP) of lymphocytes. This experimental model was also utilised to study the behaviour of T cells with dual-specific T cell receptors (TCR) during the proliferation. The Treg phenotype was studied by flow cytometry and relative gene expression with real-time polymerase chain reaction. TCR repertoires of the Tregs isolated from APECED patients and healthy controls were also compared. The dual-specific TCRs were studied with the TCR repertoire analysis that was followed with sequencing of the chosen TCR genes in order to estimate changes in the dual-specific TCR diversity. The Treg function was tested with an in vitro suppression assay. The APECED patients had normal numbers of Tregs but the phenotype and suppressive functions of the Tregs were impaired. In order to separate Aire functions in the thymus from its yet unknown role in the secondary lymphoid organs, the phenomenon of LIP was utilised. In this setting, the lymphocytes that are adoptively transferred to a lymphopenic recipient proliferate to stimuli from self-originating antigens. This proliferation can result in autoimmunity if peripheral tolerance is not fully functional. When lymphocytes that had matured without Aire in the thymus were transferred to lymphopenic Aire-sufficient recipients, no clinical autoimmunity followed. The Aire-deficient donor-originating lymphocytes hyperproliferated, and other signs of immune dysregulation were also found in the recipients. Overt autoimmunity, however, was prevented by the Aire-deficient donor-originating Tregs that hyperproliferated in the recipients. Aire-deficient lymphopenic mice were used to study whether peripheral loss of Aire had an impact on the maintenance of peripheral tolerance. When normal lymphocytes were transferred to these Aire-deficient lymphopenic recipients, the majority of recipients developed a clinically symptomatic colitis. The colitis was confirmed also by histological analysis of the colon tissue sections. In the Aire-deficient lymphopenic recipients Tregs were proliferating significantly less than in the control group s recipients that had normal Aire expression in their secondary lymphoid organs. This study shows that Aire is not only important in the central tolerance but is also has a significant role in the maintenance of the peripheral tolerance both in mice and men. Aire expressed in the secondary lymphoid organs is involved in the functions of Tregs during an immune response. This peripheral expression appears to be relatively more important in some situations since only those lymphopenic recipients that had lost peripheral expression of Aire developed a symptomatic autoimmune disease. This AIRE-related Treg defect could be clinically important in understanding the pathogenesis of APECED.

High-risk patients and high-risk grafts in infrainguinal bypass for critical limb ischaemia

The main purpose of revascularization procedures for critical limb ischaemia (CLI) is to preserve the leg and sustain the patient s ambulatory status. Other goals are ischaemic pain relief and healing of ischaemic ulcers. Patients with CLI are usually old and have several comorbidities affecting the outcome. Revascularization for CLI is meaningless unless both life and limb are preserved. Therefore, the knowledge of both patient- and bypass-related risk factors is of paramount importance in clinical decision-making, patient selection and resource allocation. The aim of this study was to identify patient- and graft-related predictors of impaired outcome after infrainguinal bypass for CLI. The purpose was to assess the outcome of high-risk patients undergoing infrainguinal bypass and to evaluate the usefulness of specific risk scoring methods. The results of bypasses in the absence of optimal vein graft material were also evaluated, and the feasibility of the new method of scaffolding suboptimal vein grafts was assessed. The results of this study showed that renal insufficiency - not only renal failure but also moderate impairment in renal function - seems to be a significant risk factor for both limb loss and death after infrainguinal bypass in patients with CLI. Low estimated GFR (PIENEMPI KUIN 30 ml/min/1.73 m2) is a strong independent marker of poor prognosis. Furthermore, estimated GFR is a more accurate predictor of survival and leg salvage after infrainguinal bypass in CLI patients than serum creatinine level alone. We also found out that the life expectancy of octogenarians with CLI is short. In this patient group endovascular revascularization is associated with a better outcome than bypass in terms of survival, leg salvage and amputation-free survival especially in presence of coronary artery disease. This study was the first one to demonstrate that Finnvasc and modified Prevent III risk scoring methods both predict the long-term outcome of patients undergoing both surgical and endovascular infrainguinal revascularization for CLI. Both risk scoring methods are easy to use and might be helpful in clinical practice as an aid in preoperative patient selection and decision-making. Similarly than in previous studies, we found out that a single-segment great saphenous vein graft is superior to any other autologous vein graft in terms of mid-term patency and leg salvage. However, if optimal vein graft is lacking, arm vein conduits are superior to prosthetic grafts especially in infrapopliteal bypasses for CLI. We studied also the new method of scaffolding suboptimal quality vein grafts and found out that this method may enable the use of vein grafts of compromised quality otherwise unsuitable for bypass grafting. The remarkable finding was that patients with the combination of high operative risk due to severe comorbidities and risk graft have extremely poor survival, suggesting that only relatively fit patients should undergo complex bypasses with risk grafts. The results of this study can be used in clinical practice as an aid in preoperative patient selection and decision-making. In the future, the need of vascular surgery will increase significantly as the elderly and diabetic population increases, which emphasises the importance of focusing on those patients that will gain benefit from infrainguinal bypass. Therefore, the individual risk of the patient, ambulatory status, outcome expectations, the risk of bypass procedure as well as technical factors such as the suitability of outflow anatomy and the available vein material should all be assessed and taken into consideration when deciding on the best revascularization strategy.