Regulations of SOX9 activity during chondrogenesis

Sox9 is a transcription factor required for chondrocyte differentiation and cartilage formation. In an effort to identify SOX9 interacting protein(s), we screened a chondrocyte cDNA library with a modified yeast two-hybrid method, Son of Sevenless (SOS) recruitment system (SRS). The catalytic subunit of cyclic AMP-dependent protein kinase A (PKA-Cα) and a new long form of c-Maf transcription factor (Lc-Maf) were found to interact specifically with SOX9. We showed here that two PKA phosphorylation consensus sites of SOX9 could be phosphorylated by PKA in vitro as well as in vivo. PKA phosphorylation of SOX9 increases its DNA binding and transcriptional activities on a Col2a1 chondrocyte-specific enhancer. Mutations of these two PKA phosphorylation sites markedly decreased the activation of SOX9 by PKA. ^ To test whether parathyroid hormone-related peptide (PTHrP) signaling results in SOX9 phosphorylation, we generated a phosphospecific antibody that specifically recognizes SOX9 that is phosphorylated at serine 181 (S 181) one of the two consensus PKA phosphorylation sites. Addition of PTHrP to COS7 cells cotransfected with SOX9 and PTH/PTHrP receptor strongly increased phosphorylation of SOX9 at S181; this phosphorylation was blocked by a PKA-specific inhibitor. In similar experiments we showed that PTHrP increased the activity of a SOX9-dependent Col2a1 enhancer. This increase in activity was abolished when a SOX9 mutant was used containing serine-to-alanine substitution in the two consensus PKA phosphorylation sites of SOX9. Using our phosphospecific SOX9 antibody we showed by immunohistochemistry of mouse embryos that Sox9 phosphorylated at S181 was localized almost exclusively in the pre-hypertrophic zone of the growth plate, an area corresponding to the major site of expression of PTH/PTHrP receptor. In contrast, no phosphorylation of Sox9 at S181 was detected in growth plates of PTH/PTHrP receptor null mutant mice. Sox9, regardless of phosphorylation state, was present in all chondrocytes of both genotypes except in hypertrophic chondrocytes. Thus, Sox9 is a target of PTHrP signaling and the PTHrP-dependent phosphorylation of SOX9 enhances its transcriptional activity. ^ In order to investigate the in vivo function of Sox9 phosphorylation by PKA, we are generating a mouse model of mutant Sox9 harboring point mutations in two PKA phosphorylation sites. Preliminary results indicated that heterozygous mice containing half amount of mutant Sox9 that can not be phosphorylated by PKA have normal skeletal phenotype and homozygous mice are being generated. ^ Lc-Maf encodes an extra ten amino acids at the carboxyl terminus of c-Maf and contains a completely different 3′ untranslated region. The interaction between SOX9 and Lc-Maf was further confirmed by co-immunoprecipitation and GST-pull down assays, which mapped the interacting domains of SOX9 to HMG DNA binding domain and that of Lc-Maf to basic leusine zipper motif. In situ hybridizations showed that RNA of Lc-Maf coexpressed with those of Sox9 and Col2a1 in areas of mesenchymal condensation during the early stages of mouse embryo development. A DNA binding site of Lc-Maf was identified at the 5′ part of a 48-bp Col2a1 enhancer element near the HMG binding site of SOX9. Lc-Maf and SOX9 synergistically activated a luciferase reporter plasmid containing a Col2al enhancer and increased the transcription of endogenous Col2a1 gene. In summary, Lc-Maf is the first identified SOX9-interating protein during chondrogenesis and may be an important activator of Col2a1 gene. ^

Mechanism of translationally coupled mRNA turnover mediated by c-fos protein-coding-region determinant

Proto-oncogene c-fos is a member of the class of early-response genes whose transient expression plays a crucial role in cell proliferation, differentiation, and apoptosis. Degradation of c- fos mRNA is an important mechanism for controlling c-fos expression. Rapid mRNA turnover mediated by the protein-coding-region determinant (mCRD) of the c-fos transcript illustrates a functional interplay between mRNA turnover and translation that coordinately influences the fate of cytoplasmic mRNA. It is suggested that mCRD communicates with the 3′ poly(A) tail via an mRNP complex comprising mCRD-associated proteins, which prevents deadenylation in the absence of translation. Ribosome transit as a result of translation is required to alter the conformation of the mRNP complex, thereby eliciting accelerated deadenylation and mRNA decay. To gain further insight into the mechanism of mCRD-mediated mRNA turnover, Unr was identified as an mCRD-binding protein, and its binding site within mCRD was characterized. Moreover, the functional role for Unr in mRNA decay was demonstrated. The result showed that elevation of Unr protein level in the cytoplasm led to inhibition of mRNA destabilization by mCRD. In addition, GST pull-down assay and immuno-precipitation analysis revealed that Unr interacted with PABP in an RNA-independent manner, which identified Unr as a novel PABP-interacting protein. Furthermore, the Unr interacting domain in PABP was characterized. In vivo mRNA decay experiments demonstrated a role for Unr-PABP interaction in mCRD-mediated mRNA decay. In conclusion, the findings of this study provide the first evidence that Unr plays a key role in mCRD-mediated mRNA decay. It is proposed that Unr is recruited by mCRD to initiate the formation of a dynamic mRNP complex for communicating with poly(A) tail through PABP. This unique mRNP complex may couple translation to mRNA decay, and perhaps to recruit the responsible nuclease for deadenylation. ^

A role for p53 in sensing DNA damage and triggering apoptotic responses to anticancer agents

The p53 tumor suppressor protein plays a major role in cellular responses to anticancer agents that target DNA. DNA damage triggers the accumulation of p53, resulting in the transactivation of genes, which induce cell cycle arrest to allow for repair of the damaged DNA, or signal apoptosis. The exact role that p53 plays in sensing DNA damage and the functional consequences remain to be investigated. The main goal of this project was to determine if p53 is directly involved in sensing DNA damage induced by anticancer agents and in mediating down-stream cellular responses. This was tested in two experimental models of DNA damage: (1) DNA strand termination caused by anticancer nucleoside analogs and (2) oxidative DNA damage induced by reactive oxygen species (ROS). Mobility shift assays demonstrated that p53 and DNA-PK/Ku form a complex that binds DNA containing the anticancer nucleoside analog gemcitabine monophosphate in vitro. Binding of the p53-DNA-PK/Ku complex to the analog-containing DNA inhibited DNA strand elongation. Furthermore, treatment of cells with gemcitabine resulted in the induction of apoptosis, which was associated with the accumulation of p53 protein, its phosphorylation, and nuclear localization, suggesting the activation of p53 to trigger apoptosis following gemcitabine induced DNA strand termination. The role of p53 as a DNA damage sensor was further demonstrated in response to oxidative DNA damage. Protein pull-down assays demonstrated that p53 complexes with OGG1 and APE, and binds DNA containing the oxidized DNA base 8-oxoG. Importantly, p53 enhances the activities of APE and OGG1 in excising the 8-oxoG residue as shown by functional assays in vitro. This correlated with the more rapid removal of 8-oxoG from DNA in intact cells with wild-type p53 exposed to exogenous ROS stress. Interestingly, persistent exposure to ROS resulted in the accelerated onset of apoptosis in cells with wild-type p53 when compared to isogenic cells lacking p53. Apoptosis in p53+/+ cells was associated with accumulation and phosphorylation of p53 and its nuclear localization. Taken together, these results indicate that p53 plays a key role in sensing DNA damage induced by anticancer nucleoside analogs and ROS, and in triggering down-stream apoptotic responses. This study provides new mechanistic insights into the functions of p53 in cellular responses to anticancer agents. ^

The University Compost Facility after Three Years

The University Compost Facility, 52274 260th St., Ames, Iowa has completed three full years of operation. The facility is managed by the ISU Research Farms and has a separate revolving account that receives fees and sales, and pays expenses. The facility is designed to be self-supporting, i.e. not receive allocations for its operations. The facility consists of seven, 80 × 140 ft hoop barns and a new 55 × 120 ft hoop barn, all with paved floors. The facility also has a Mettler-Toledo electronic scale with a 10 ft × 70 ft platform to weigh all materials. Key machinery is 1) compost turner, a used pull-type Aeromaster PT-170, 14 ft wide made by Midwest Biosystems, Tampico, IL; 2) a converted dump truck trailer used to construct windrows and haul material; 3) telehandler, Caterpillar TH407 with cab and 2.75 cubic yard bucket; and 4) tractor, John Deere 7520 (125 hp) with IVT (Infinite Variable Transmission) and front-wheel assist used to pull the turner and dump trailer.

Sedimentology, magnetics and chemistry on palaeolake core D100 from the Gobi Desert

The Gaxun Nur Basin in arid China is tectonically influenced by the left-lateral displacements along the Gobi-Altay and Qilian Shan shear zones, resulting in a large pull-apart basin with strong subsidence in the interior. The up to 300 m thick basin fills consist of fluvio-lacustrine fine-grained deposits mainly transported by river discharges from the Tibetan Plateau. They led to a large depositional area of more than 28,000 qkm in size with presently dry terminal lakes at the outer edges. This vast area serves as a main source for loess transport to south-eastern regions of China (Loess Plateau) caused by the variable winter monsoon. Based on geochemical and sedimentological analyses of the sediment core D100 retrieved from a deep drilling in the centre of the Gaxun Nur Basin following questions have to be answered: 1. Reconstruction of the water balance and determination of hydrological cycles during interglacial and glacial periods. 2. Reconstructing variations in lacustrine environment and aeolian activities with respect to transitional phases fro, warm to cold stages (MIS 4 to 5 and older stages). 3. Establishing a sustainable chronology for the last 250 ka.

Mineralogy and structural specification of ODP Leg 125 peridotite samples

Abundant serpentinite seamounts are found along the outer high of the Mariana forearc at the top of the inner slope of the trench. One of them, Conical Seamount, was drilled at Sites 778, 779, and 780 during Leg 125. The rocks recovered at Holes 779A and 780C, respectively, on the flanks and at the summit of the seamount, include moderately serpentinized depleted harzburgites and some dunites. These rocks exhibit evidence of resorption of the orthopyroxene, when present, and the local presence of very calcic-rich diopside in veins oblique to the main high-temperature foliation of the rock. The peridotites, initially well-foliated with locally poikiloblastic textures, show overprints of a two-stage deformation history: (1) a high-temperature (>1000°C), low-stress (0.02 GPa), homogeneous deformation that has led to the present Porphyroclastic textures displayed by the rocks and (2) heterogeneous ductile shearing at a much higher stress (0.05 GPa). This heterogeneous shearing probably describes a single tectonic event because it began at high temperatures, producing dynamic recrystallization of olivine in the shear zone, and ended at low temperatures in the stability field of chlorite and serpentine. In a few samples, olivine shows evidence of quasi-hydrostatic recrystallization at a very high temperature. Here, we propose that this recrystallization was related to fluid/magma percolation, a process that can also account for the resorption of the orthopyroxene and for the late crystallization of diopside veins in the rock. The impregnation by fluid or magma, development of the main high-temperature, low-stress deformation, and subsequent migration recrystallization of olivine probably occurred in a mantle fragment involved in the arc formation. In addition, this mantle has preserved structures that may have formed earlier in the oceanic lithosphere upon which the arc formed. Heterogeneous ductile shear zones in the peridotites may have developed during uplift. The "cold" deformation may have taken place during diapiric rise of hot mantle that underwent subsequent serpentinization or gliding along normal faults associated with the extension of the eastern margin of the forearc.

Composition of volcanic rocks from the Alchan Basin, Northwestern Primorie

Geological, petrochemical, and geochemical data are reported for volcanic rocks of a Cretaceous pull-apart basin in the Tan Lu strike-slip system, Asian continental margin. A comparison of these volcanic rocks with magmatic rocks from typical Cenozoic transform margins in the western North America and rift zones of Korea made it possible to distinguish some indicator features of transform-margin volcanic rocks. Magmatic rocks from strike-slip extension zones bear island-arc, intraplate, and occasionally depleted MORB geochemical signatures. In addition to calc-alkaline rocks there are bimodal volcanic series. The rocks are characterized by high K2O, MgO, and TiO2 contents. They show variable enrichment in LILE relative to HFSE, which is typical of island-arc magmas. At the same time they are rich in compatible transition elements, which is a characteristic of intraplate magmas. Trace element distribution patterns normalized to MORB or primitive mantle usually show a negative Ta-Nb anomaly typical of suprasubduction settings. Their Ta/Nb ratio is lower, whereas Ba/Nb, Ba/La, and La/Yb ratios are higher than those of some MORB and OIB. In terms of trace element systematics, for example, Ta-Th-Hf, Ba/La-(Ba/La)_n, (La/Sm)_n-La/Hf, and others, they fall within the area of mixing of magmas from several sources (island arc, intraplate, and depleted reservoirs). Magmatic rocks of transform settings show a sigmoidal chondrite-normalized REE distribution pattern with a negative slope of LREE, depletion in MREE, and an enriched or flat HREE pattern. Magmas with mixed geochemical characteristics presumably originated in a transform margin setting in local extension zones under influence of mantle diapirs, which caused metasomatism and melting of the lithosphere at different levels, and mixing of melts from different sources in variable proportions.

Pore depth evolution of a pyrochlore pellet

The long-term stability of ceramic materials that are considered as potential nuclear waste forms is governed by heterogeneous surface reactivity. Thus, instead of a mean rate, the identification of one or more dominant contributors to the overall dissolution rate is the key to predict the stability of waste forms quantitatively. Direct surface measurements by vertical scanning interferometry (VSI) and their analysis via material flux maps and resulting dissolution rate spectra provide data about dominant rate contributors and their variability over time. Using pyrochlore (Nd2Zr2O7) pellet dissolution under acidic conditions as an example, we demonstrate the identification and quantification of dissolution rate contributors, based on VSI data and rate spectrum analysis. Heterogeneous surface alteration of pyrochlore varies by a factor of about 5 and additional material loss by chemo-mechanical grain pull-out within the uppermost grain layer. We identified four different rate contributors that are responsible for the observed dissolution rate range of single grains. Our new concept offers the opportunity to increase our mechanistic understanding and to predict quantitatively the alteration of ceramic waste forms.

Rural to Urban Migration: A District Level Analysis for India

Based on the recent census data this paper analyses the district level rural to urban migration rates (both intra-state and the inter-state) among males and females separately. Both the rates are closely associated irrespective of whether the migrants originate from the rural areas within the state or outside the state. This would suggest that women usually migrate as accompanists of the males. Though many of the relatively poor and backward states actually show large population mobility, which is primarily in search of a livelihood, the mobility of male population is also seen to be prominent in the relatively advanced states like Maharashtra and Gujarat. Rapid migration of rural females within the boundaries of the states is, however, evident across most of the regions. The social networks, which play an important role in the context of migration are prevalent among the short distance migrants and tend to lose their significance with a rise in the distance between the place of origin and destination though there are some exceptions to this phenomenon. Besides the north-south divide in the Indian context is indeed a significant phenomenon with a few exceptions of metropolitan cities. As regards the effect of factors at the place of destination, prospects for better job opportunities are a major determinant of male migration. Low castes and minority groups tend to pull migration through network effects. Among females also these effects are evident though with the inclusion of the male migration rate they become less significant. Finally the paper brings out the policy implications.

Análisis de los modelos de comportamiento de vigas de hormigón armado reforzadas a cortante con polímeros armados con fibras (frp). Validación y calibración experimental

Los polímeros armados con fibras (FRP) se utilizan en refuerzos de estructuras de hormigón debido sobre todo a sus excelentes propiedades mecánicas, su resistencia a la corrosión y a su ligereza que se traduce en facilidad y ahorro en el transporte, puesta en obra y aplicación, la cual se realiza de forma muy rápida, con pocos operarios y utilizando medios auxiliares ligeros, minimizándose las interrupciones del uso de la estructura y las molestias a los usuarios. Las razones presentadas anteriormente, han despertado un gran inter´es por parte de diferentes grupos de investigación a nivel mundial y que actualmente se encuentran desarrollando nuevas técnicas de aplicación y métodos de cálculo. Sin embargo, las investigaciones realizadas hasta la fecha, muestran un procedimiento bien definido y aceptado en lo referente al cálculo a flexión, lo cual no ocurre con el refuerzo a cortante y aunque se ha demostrado que el refuerzo con FRP es un sistema eficaz para incrementar la capacidad ´ultima frente a esfuerzos cortantes, también se pone de manifiesto la necesidad de más estudios experimentales y teóricos para avanzar en el entendimiento de los mecanismos involucrados para este tipo de refuerzo y establecer un procedimiento de diseño apropiado que maximice las excelentes propiedades de este material. Los modelos que explican el comportamiento del refuerzo a cortante de elementos de hormigón armado son complejos y sin transposición directa a fórmulas ingenieriles. Las normas actualmente en vigor, generalmente, establecen empíricamente la capacidad cortante como la suma de las capacidades del hormigón y el refuerzo transversal de acero. Cuando un elemento es reforzado externamente con FRP, los modelos son evidentemente aun más complejos. Las guías y recomendaciones existentes proponen calcular la capacidad del elemento añadiendo la resistencia aportada por el refuerzo externo de FRP a la ya dada por el hormigón y acero transversal. Sin embargo, la idoneidad de este acercamiento es cuestionable puesto que no tiene en cuenta una posible interacción entre refuerzos. Con base en lo anterior se da origen al tema objeto de este trabajo, el cual está orientado al estudio a cortante de elementos de hormigón armado (HA), reforzados externamente con material compuesto de tejido unidireccional de fibra de carbono y resina epoxi. Inicialmente se hace una completa revisión del estado actual del conocimiento de la resistencia a cortante en elementos de hormigón armado con y sin refuerzo externo de FRP, prestando especial atención en los mecanismos actuantes estudiados hasta la fecha. La bibliografía consultada ha sido exhaustiva y actualizada lo que ha permitido el estudio de los modelos propuestos más importantes, tanto para la descripción del fenómeno de adherencia entre hormigón-FRP como de la valoración del aporte al cortante total hecho por el FRP, a través de sendas bases de datos de ensayos de pull-out y de vigas de hormigón armado ensayadas a cortante. Con base en todo lo anterior, se expusieron los mecanismos actuantes en el aporte a cortante hecho por el FRP en elementos de hormigón armado y la forma como las principales guías de cálculo existentes hasta la fecha los abordan. De igual forma se define un modelo de resistencia de esfuerzos para el FRP y se proponen dos modelos para el cálculo de las tensiones o deformaciones efectivas, de los cuales uno esta basado en el modelo de adherencia propuesto por Oller (2005) y el otro en una regresión multivariante para los mecanismos expuestos. Como complemento del estudio de los trabajos encontrados en la literatura, se lleva acabo un programa experimental que, además de aportar más registros a la exigua base de datos existentes, aporte mayor luz a los puntos que se consideran están deficientemente resueltos. Dentro de este programa se realizaron 32 ensayos sobre 16 vigas de 4.5 m de longitud (dos ensayos por viga), reforzadas a cortante con tejido unidireccional de CFRP. Finalmente, estos estudios han permitido proponer modificaciones a las formulaciones existentes en los códigos y guías en vigor. Abstract Its excellent mechanical properties, as well as its corrosion resistance and light weight, which make it easy to apply and inexpensive to ship to the worksite, are the basis of the extended use of fiber reinforced polymer (FRP) as external strengthening for structures. FRP strengthening is a rapid operation calling for only limited labor and lightweight ancillary equipment, all of which minimizes both the interruption of facility usage and user inconvenience. These advantages have aroused considerable interest in civil engineering science and technology and have led to countless applications the world over. Research studies on the shear strength of FRP-strengthened members have been much fewer in number and more controversial than the research on flexural strengthening, for which a more or less standardized and generally accepted procedure has been established. The research conducted and a host of applications around the world have shown that FRP strengthening is an effective technique for raising ultimate shear strength, but it has also revealed a need for further experimental and theoretical research to advance in the understanding of the mechanisms involved and establish suitable design procedures that optimize the excellent properties of this material The models that explain reinforced concrete (RC) shear strength behavior are complex and cannot be directly transposed to engineering formulas. The standards presently in place generally establish shear capacity empirically as the sum of the capacities of the concrete and the passive reinforcement. When members are externally strengthened with FRP, the models are obviously even more complex. The existing guides and recommendations propose calculating capacity by adding the external strength provided by the FRP to the contributions of the concrete and passive reinforcement. The suitability of this approach is questionable, however, because it fails to consider the interaction between passive reinforcement and external strengthening. The subject of this work is based in above, which is focused on externally shear strengthening for reinforced concrete members with unidirectional carbon fiber sheets bonded with epoxy resin. v Initially a thorough literature review on shear of reinforced concrete beams with and without external FRP strengthening was performed, paying special attention to the acting mechanisms studied to date, which allowed the study of the most important models both to describe the bond phenomenon as well as calculating the FRP shear contribution, through separate databases of pull-out tests and shear tests on reinforced concrete beams externally strengthened with FRP. Based on above, they were exposed the acting mechanisms in a FRP shear strengthening on reinforced concrete beams and how guidelines deal the topic. The same way, it is defined a FRP stress strength model and two more models are proposed for calculating the effective stress, one of these is based on the Oller (2005) bond model and another one is the data best fit, taking into account most of the acting mechanisms. To complement the theoretical part we develop an experimental program that, in addition to providing more records to the meager existing database provide greater understanding to the points considered poorly resolved. The test program included 32 tests of 16 beams (2 per beam) of 4.5 m long, shear strengthened with FRP, externally. Finally, modifications to the existing codes and guidelines are proposed.

Amplificador de potencia clase F a 1.64 ghz con control de armónicos

This paper presents a high-power high efficiency PA design method using load pull technique. Harmonic impedance control at the virtual drain is accomplished through the use of tunable pre-matching circuits and modeling of package parasitics. A 0.5 µm GaN high electron mobility transistor (HEMT) is characterized using the method, and loadpull measurements are simulated illustrating the impact of varying 2nd and 3rd harmonic termination. These harmonic terminations are added to satisfy conditions for class-F load pull. The method is verified by design and simulation of a 40-W class-F PA prototype at 1.64 GHz with 76% drain efficiency and 10 dB gain (70% PAE).

Modelización numérica del comportamiento mecánico de un fieltro de polipropileno

En el presente trabajo se presenta un modelo del continuo para un fieltro denso. Igualando la densidad de potencia de un elemento del continuo a la densidad de potencia mecánica que actúa sobre el conjunto de las fibras se obtiene una expresión del tensor de tensiones en la configuración de referencia. El modelo se completa mediante la inclusión de un modelo de daño para modelar fenomenológicamente los mecanismos de extracción (pull-out) y rotura de las fibras. Se ha implementado el modelo como una subrutina de material de usuario para un código de elementos finitos (ABAQUSExplicit), formulado en grandes deformaciones. Los resultados obtenidos se han comparado con experimentos realizados sobre un fieltro comercial (geotextil) de fibras de polipropileno y muestran que el modelo es capaz de reproducir el comportamiento del material hasta la localización del daño y pérdida de capacidad portante del mismo.

Estudio de la capacidad de arranque de una dinamita gelatinosa frente a una dinamita de seguridad

The objective of this project is to show that the permissible explosive called 20 SR is able to pull out the coal in the normal conditions of blasting in a satisfactory way and to set up the equivalence between the 20 SR and gelatin dynamite (Goma 2 ECO). To achieve this goal some blasting were done, changing the conditions of the blasting and the powder factor for the 20 SR. To analyze the fragmentation base on the analysis of the images of the rock blasted, a commercial software was used. The results from this analysis were compared with the results from the theoretical model for fragmentation created by Kuz – Ram. After all, it was showed that the 20 SR explosive is able to pull out the coal for different coal rock compositions. As the result of this project we can conclude that the 20 SR seems to be able to pull out the coal in normal blasting conditions, using the powder factor as a proportion of the “ballistic mortar” between the two explosives.

Tractor PT: A traction prediction software for agricultural tractor

Traction prediction modelling, a key factor in farm tractor design, has been driven by the need to find the answer to this question without having to build physical prototypes. A wide range of theories and their respective algorithms can be used in such predictions. The “Tractors and Tillage” research team at the Polytechnic University of Madrid, which engages, among others, in traction prediction for farm tractors, has developed a series of programs based on the cone index as the parameter representative of the terrain. With the software introduced in the present paper, written in Visual Basic, slip can be predicted in two- and four-wheel drive tractors using any one of four models. It includes databases for tractors, front tyres, rear tyres and working conditions (soil cone index and drawbar pull exerted). The results can be exported in spreadsheet format.