IGHD II: A Novel GH-1 Gene Mutation (GH-L76P) Severely Affects GH Folding, Stability, and Secretion

CONTEXT The autosomal dominant form of GH deficiency (IGHD II) is characterized by markedly reduced GH secretion combined with low concentrations of IGF-1 leading to short stature. OBJECTIVE Structure-function analysis of a missense mutation in the GH-1 gene converting codon 76 from leucine (L) to proline (P) yielding a mutant GH-L76P peptide. DESIGN, SETTINGS, AND PATIENTS Heterozygosity for GH-L76P/wt-GH was identified in a nonconsanguineous Spanish family. The index patients, two siblings, a boy and a girl, were referred for assessment of their short stature (-3.2 and -3.8 SD). Their grandmother, father, and aunt were also carrying the same mutation and showed severe short stature; therefore, IGHD II was diagnosed. INTERVENTIONS AND RESULTS AtT-20 cells coexpressing both wt-GH and GH-L76P showed a reduced GH secretion (P < .001) after forskolin stimulation when compared with the cells expressing only wt-GH. In silico mutagenesis and molecular dynamics simulations presented alterations of correct folding and mutant stability compared with wt-GH. Therefore, further structural analysis of the GH-L76P mutant was performed using expressed and purified proteins in Escherichia coli by thermofluor assay and fast degradation proteolysis assay. Both assays revealed that the GH-L76P mutant is unstable and misfolded compared to wt-GH confirming the bioinformatic model prediction. CONCLUSIONS This is the first report of a family suffering from short stature caused by IGHD II, which severely affects intracellular GH folding and stability as well as secretion, highlighting the necessity of functional analysis of any GH variant for defining new mechanisms as a cause for IGHD II.

Heregulin -beta 1 directly associates with RhoA via the immunoglobulin -like domain thereby modulating breast cancer metastasis-related properties

Heregulins constitute a family of growth factors belonging to the epidermal growth factor (EGF) family. Breast cancers that overexpress specific members of the EGF receptor family (EGFR, ErbB2, ErbB3, ErbB4) have increased metastatic potential, and Heregulin-β1 (HRGβ1), a ligand for ErbB3 and ErbB4, has also been shown to induce metastasis-related properties in breast cancer cells in vitro. The secreted form of the HRGβ1 is composed of five distinct structural domains, including the N-terminal domain, an immunoglobulin-like domain (IgG-like), a glycosylation domain, an EGF-like domain, and a β1-specific domain. Of these, the EGF-like domain is well characterized for its function in metastasis-related properties as well as its structure. However, the contributions of the other HRGβ1 domains in breast cancer metastasis remains unclear. ^ To investigate this, HRGβ1 proteins with targeted domain deletions were purified and subjected to assays for metastasis-related properties, including aggregation, invasion, activation of EGFR family members, and motility of breast cancer cells. These assays showed that retaining the EGF-like domain of HRGβ1 is important for activation of EGFRs. Interestingly, the HRGβ1 protein lacking the IgG-like domain (NGEB) led to a decrease in breast cancer cell motility, indicating the IgG-like domain modulates cell motility, an important step in cancer metastasis. ^ To understand the underlying mechanisms, I performed protein sequence and structural analysis of HRGβ1 and identified that the IgG-like domain of HRGβ1 shares sequence homology and three-dimensional structural similarity with the IgG-like domain of TRIO. TRIO is a cytoplasmic protein that directly associates with RhoA, a GTPase involved in cell reorganization and cell motility. Therefore, I hypothesized that HRGβ1 may translocate inside the breast cancer cells through receptor mediated endocytosis and bind to RhoA via its IgG-like domain. I show wild type HRGβ1 but not NGEB binds RhoA in vitro and in vivo, leading to RhoA activation. Inhibition of HRG-β1 internalization via endocytosis disrupted HRGβ1 binding to RhoA. Additionally, breast cancer cell motility induced by HRG-β1 is reduced after treatment with inhibitors to both endocytosis and RhoA function, similar to levels seen with NGEB treatment. ^ Thus, in addition to the well-known role of HRGβ1 as an extracellular stimulator of the EGFR family members, HRGβ1 also functions within the cell as a binding partner and activator of RhoA to modulate cancer cell motility. ^

(Table 1) Description of structures in cores at DSDP Holes 59-451, 59-448A and 59-447A

Fifteen lengths of Leg 59 cores (primarily from Hole 451 as well as from Holes 447A and 448A) exhibiting macroscopic faults were selected by Dr. R. B. Scott (Co-Chief Scientist, Leg 59) to help us initiate this petrofabric analysis. We proposed to (1) determine what dynamically useful deformation features might be associated with the faults, and (2) infer from these features as much as possible about the physical environment of the deformation (effective pressure, differential stress, temperature, and strain rate), the orientation and relatively magnitudes of the principal stresses at the time of deformation, and the degree of induration of the rocks at the time of deformation. The cores, mainly from Hole 451, had been slabbed on board ship with respect to the trace of bedding so that each cut surface contains the true bedding dip-direction. In general, the cores from Hole 451 are largely calcareous, lithic and vitric, brecciated tuffs, whereas those from Holes 447A and 448A are basalts or basalt breccias.

Summary of physical properties on cores used for petrofabric analyses triaxial experiments at DSDP Leg 67 Holes

Deformation features within the cores are studied with a view towards elucidating the structure of the Middle America Trench along the transect drilled during Leg 67. Where possible, inferences are made as to the physical environment of deformation. Extensional tectonics prevails in the area of the seaward slope and trench. Fracturing and one well-preserved normal fault are found mostly within the lower Miocene chalks, at the base of the sedimentary section. These chalks have high porosities (40%-60%) and water content (30%-190%, based on % dry wt.). Experimental triaxial compression tests conducted on both dry and water-saturated samples of chalk from Holes 495 and 499B show that only in the saturated samples is more brittle behavior observed. Brittle failure of the chalks is greatly facilitated by pore fluid pressures that lead to low effective pressures. Additional embrittlement (weakening) can take place as a result of the imposed extensional stress resulting from bending of a subducting elastic oceanic plate. The chalks exhibit, in a landward direction, an increase in density and mechanical strength and a decrease in water content. These changes are attributed to mechanical compaction that may have resulted from tectonic horizontal compression. The structure of the landward slope is not well understood because the slope sites had to be abandoned due to the presence of gas hydrate. The relationship of the chaotic, brittle deformation (observed in the cores from Hole 494A) at the base of the landward slope to tectonic processes remains unclear. The deformation observed on the slope sites (Holes 496 and 497) is mostly fracturing and near-vertical sigmoidal veinlets. These are interpreted as being the result of gas/fluid overpressurization due to the decomposition of the gas hydrate, and not due to tectonic loading of accreted sediments. Aside from four small displacement (less than 1cm) reverse faults observed in the lower Miocene chalks (which may be the product of soft-sediment deformation), there is a noticeable absence of structures reflecting a dominance of horizontal (tectonic) compression along the transect drilled. The absence of such features, the lack of continuity of sediment types across the trench-landward slope, and the normal stratigraphic sequence in Hole 494A do not support any known accretionary model.

Geochemistry of samples from Valtournenche, Western Italian Alps

Slices of polycyclic metasediments (marbles and meta-cherts) are tectonically amalgamated with the polydeformed basement of the Dent Blanche tectonic system along a major Alpine shear zone in the Western Alps (Becca di Salé area, Valtournenche Valley). A combination of techniques (structural analysis at various scales, metamorphic petrology, geochronology and trace element geochemistry) was applied to determine the age and composition of accessory phases (titanite, allanite and zircon) and their relation to major minerals. The results are used to reconstruct the polyphase structural and metamorphic history, comprising both pre-Alpine and Alpine cycles. The pre-Alpine evolution is associated with low-pressure high-temperature metamorphism related to Permo-Triassic lithospheric thinning. In meta-cherts, microtextural relations indicate coeval growth of allanite and garnet during this stage, at ~ 300 Ma. Textures of zircon also indicate crystallization at HT conditions; ages scatter from 263-294 Ma, with a major cluster of data at ~ 276 Ma. In impure marble, U-Pb analyses of titanite domains (with variable Al and F contents) yield apparent 206Pb/238U dates range from Permian to Jurassic. Chemical and isotopic data suggest that titanite formed at Permian times and was then affected by (extension-related?) fluid circulation during the Triassic and Jurassic, which redistributed major elements (Al and F) and partially opened the U-Pb system. The Alpine cycle lead to early blueschist facies assemblages, which were partly overprinted under greenschist facies conditions. The strong Alpine compressional overprint disrupted the pre-Alpine structural imprint and/or reactivated earlier structures. The pre-Alpine metamorphic record, preserved in these slices of metasediments, reflects the onset of the Permo-Triassic lithospheric extension to Jurassic rifting.

Preliminary Study and Proposal for Restoring a Jesuit College at Saragossa, Spain

A historical study and a construction pathology survey were conducted prior to proposing a solution for restoring the upper windows on the south side of Old San Carlos College at Saragossa (16th-17th centuries), wich had been forteited to add a third storey to the cloister. Althought initially designed to a simple large hall format, the church is a harmonius blend of Aragonese Gothic architecture and typical Jesuit scheme, consisting in a central nave flanket by chapel-confessional and raised galleries for the community. A subsequent enlargement of the roof, wich rests on the original framing over the central nave, reduced the mechanical strength of the principal rafters on the opposite side, prompting a concomitant imbalance of forces that has affected the entire structure. In view of the foregoing, in addition to restituting the upper window, the proposal solution envisages restoring the roof over the central nave to its original design, and with the interior lighting in the church.

Shower approach in the simulation of ion scattering from solids

An efficient approach for the simulation of ion scattering from solids is proposed. For every encountered atom, we take multiple samples of its thermal displacements among those which result in scattering with high probability to finally reach the detector. As a result, the detector is illuminated by intensive “showers,” where each event of detection must be weighted according to the actual probability of the atom displacement. The computational cost of such simulation is orders of magnitude lower than in the direct approach, and a comprehensive analysis of multiple and plural scattering effects becomes possible. We use this method for two purposes. First, the accuracy of the approximate approaches, developed mainly for ion-beam structural analysis, is verified. Second, the possibility to reproduce a wide class of experimental conditions is used to analyze some basic features of ion-solid collisions: the role of double violent collisions in low-energy ion scattering; the origin of the “surface peak” in scattering from amorphous samples; the low-energy tail in the energy spectra of scattered medium-energy ions due to plural scattering; and the degradation of blocking patterns in two-dimensional angular distributions with increasing depth of scattering. As an example of simulation for ions of MeV energies, we verify the time reversibility for channeling and blocking of 1-MeV protons in a W crystal. The possibilities of analysis that our approach offers may be very useful for various applications, in particular, for structural analysis with atomic resolution.

Obtaining carbonation depth of concrete used two different procedures standard EHE-08

In the Standard EHE 08, for the first time, durability acquires the status of Limit State. Article 8 provides that the term Durability limit state, produced by physical and chemical actions, different loads and actions of structural analysis, which can degrade the concrete and reinforcement to unacceptable limits. The verification of this limit state can be done through a procedure set out in the provisions of the Standard. This procedure is based on the use of tables that, depending on the aggressiveness of the environment in which the structure is the concrete strength and the life of the project, setting the quality of the concrete cover (minimum thickness and maximum water cement ratio of concrete used) and the maximum crack width. This procedure, simple in its application, provides highly secure solutions. In addition, on Annex 9, the Standard EHE 08 offers models for testing the durability limit state in cases of corrosion of reinforcement due to carbonation of concrete or entry of chloride ions. The results obtained with these models are tighter than those obtained with the procedure of the articles. In this paper we use both methods in the study of reinforced concrete structures with potential problems of corrosion of reinforcement due to carbonation of concrete. Later checking the results obtained by both procedures. Results demonstrate that the use of the models listed in Annex 9 of Standard EHE 08 offer cheaper solutions than those obtained using the procedure of the articles

Wedges and plate-bandes: mechanical theories after De la Hire

Plate-bandes are straight masonry arches (they are called, also, flat arches or lintel arches). Ideally they have the surfaces of extrados and intrados plane and horizontal. The stones or bricks have radial joints converging usually in one centre. The voussoirs have the form of wedges and in French they are called "claveaux". A plate-bande is, in fact, a lintel made of several stones and the proportions of lintels and plate-bandes are similar. Proportions of plate-bandes, that is the relationship between the thickness t and the span s (t/s)varies, typically between 1/4–1/3 in thick plate-bandes, and is less than 1/20 in the most slender ones. A ratio of circa 1/8 was usual in the 18th Century and follows a simple geometrical rule: the centre form with the intrados an equilateral triangle and the plate-bande should contain an arc of circle. The joints are usually plane, but in some cases present a «rebated» or «stepped» form. Plate-bandes exert an inclined thrust as any masonry arch. This thrust is usually very high and it requires either massive buttresses, or to be built in the middle of thick walls. Master builders and architects have tried since antiquity to calculate the abutment necessary for any arch. A modern architect or engineer will measure the arch thrust in units of force, kN or tons. Traditionally, the thrust has been measured as the size of the buttresses to resist it safely. Old structural rules, then, addressed the design problem establishing a relationship between the span and the depth of the buttress. These were empirical rules, particular for every type of arch or structure in every epoch. Thus, the typical gothic buttress is 1/4 of the vault span, but a Renaissance or baroque barrel vault will need more than 1/3 of the span. A plate-bande would require more than one half of the span; this is precisely the rule cited by the French engineer Gautier, who tried unsuccessfully to justify it by static reasons. They were used, typically, to form the lintels of windows or doors (1-2 m, typically); in Antiquity they were used, also, though rarely, at the gates of city walls or in niches (ca. 2 m, reaching 5.2 m). Plate-bandes may show particular problems: it is not unusual that some sliding of the voussoirs can be observed, particularly in thick plate-bandes. The stepped joints on Fig. 1, left, were used to avoid this problem. There are other «hidden» methods, like iron cramps or the use of stone wedges, etc. In seismic zones these devices were usual. Another problem relates to the deformation; a slight yielding of the abutments, or even the compression of the mortar joints, may lead to some cracking and the descent of the central keystone. Even a tiny descent will convert the original straight line of the intrados in a broken line with a visible «kink» or angle in the middle. Of course, both problems should be avoided. Finally, the wedge form of the voussoirs lead to acute angles in the stones and this can produce partial fractures; this occurs usually at the inferior border of the springers at the abutments. It follows, that to build a successful plate-bande is not an easy matter. Also, the structural study of plate-bandes is far from simple, and mechanics and geometry are related in a particular way. In the present paper we will concentrate on the structural aspects and their constructive consequences, with a historical approach. We will outline the development of structural analysis of plate-bandes from ca. 1700 until today. This brief history has a more than purely academic interest. Different approaches and theories pointed to particular problem, and though the solution given may have been incorrect, the question posed was often pertinent. The paper ends with the application of modern Limit Analysis of Masonry Structures, developed mainly by professor Heyman in the last fifty years. The work aims, also, to give some clues for the actual architect and engineer involved in the analysis or restoration of masonry buildings.

Problemas de interacción suelo estructura en cimentaciones y muros de contención. Influencia de los fenómenos de despegue y deslizamiento

Los terremotos constituyen una de las más importantes fuentes productoras de cargas dinámicas que actúan sobre las estructuras y sus cimentaciones. Cuando se produce un terremoto la energía liberada genera movimientos del terreno en forma de ondas sísmicas que pueden provocar asientos en las cimentaciones de los edificios, empujes sobre los muros de contención, vuelco de las estructuras y el suelo puede licuar perdiendo su capacidad de soporte. Los efectos de los terremotos en estructuras constituyen unos de los aspectos que involucran por su condición de interacción sueloestructura, disciplinas diversas como el Análisis Estructural, la Mecánica de Suelo y la Ingeniería Sísmica. Uno de los aspectos que han sido poco estudiados en el cálculo de estructuras sometidas a la acciones de los terremotos son los efectos del comportamiento no lineal del suelo y de los movimientos que pueden producirse bajo la acción de cargas sísmicas, tales como posibles despegues y deslizamientos. En esta Tesis se estudian primero los empujes sísmicos y posibles deslizamientos de muros de contención y se comparan las predicciones de distintos tipos de cálculos: métodos pseudo-estáticos como el de Mononobe-Okabe (1929) con la contribución de Whitman-Liao (1985), y formulaciones analíticas como la desarrollada por Veletsos y Younan (1994). En segundo lugar se estudia el efecto del comportamiento no lineal del terreno en las rigideces de una losa de cimentación superficial y circular, como la correspondiente a la chimenea de una Central Térmica o al edificio del reactor de una Central Nuclear, considerando su variación con frecuencia y con el nivel de cargas. Finalmente se estudian los posibles deslizamientos y separación de las losas de estas dos estructuras bajo la acción de terremotos, siguiendo la formulación propuesta por Wolf (1988). Para estos estudios se han desarrollado una serie de programas específicos (MUROSIS, VELETSOS, INTESES y SEPARSE) cuyos listados y detalles se incluyen en los Apéndices. En el capítulo 6 se incluyen las conclusiones resultantes de estos estudios y recomendaciones para futuras investigaciones. ABSTRACT Earthquakes constitute one of the most important sources of dynamic loads that acting on structures and foundations. When an earthquake occurs the liberated energy generates seismic waves that can give rise to structural vibrations, settlements of the foundations of buildings, pressures on retaining walls, and possible sliding, uplifting or even overturning of structures. The soil can also liquefy losing its capacity of support The study of the effects of earthquakes on structures involve the use of diverse disciplines such as Structural Analysis, Soil Mechanics and Earthquake Engineering. Some aspects that have been the subject of limited research in relation to the behavior of structures subjected to earthquakes are the effects of nonlinear soil behavior and geometric nonlinearities such as sliding and uplifting of foundations. This Thesis starts with the study of the seismic pressures and potential displacements of retaining walls comparing the predictions of two types of formulations and assessing their range of applicability and limitations: pseudo-static methods as proposed by Mononobe-Okabe (1929), with the contribution of Whitman-Liao (1985), and analytical formulations as the one developed by Veletsos and Younan (1994) for rigid walls. The Thesis deals next with the effects of nonlinear soil behavior on the dynamic stiffness of circular mat foundations like the chimney of a Thermal Power Station or the reactor building of a Nuclear Power Plant, as a function of frequency and level of forces. Finally the seismic response of these two structures accounting for the potential sliding and uplifting of the foundation under a given earthquake are studied, following an approach suggested by Wolf (1988). In order to carry out these studies a number of special purposes computer programs were developed (MUROSIS, VELETSOS, INTESES and SEPARSE). The listing and details of these programs are included in the appendices. The conclusions derived from these studies and recommendations for future work are presented in Chapter 6.