Bone loss in KLHL3 knock-in mice characterized by a pseudohypoaldosteronism type II-like phenotype is mediated by renal PTH resistance

This is the final versionPoster presented at the 43rd Annual European Calcified Tissue Society Congress, Rome, Italy, 14 - 17 May 2016Pseudohypoaldosteronism type II (PHAII) is a hereditary disease characterized by hypertension, hypercalciuria and osteopenia. PHAII is caused by mutations in with-no-lysine kinase 1 (WNK1), WNK4, or the cullin RING ligase family members kelch-like 3 (KLHL3) or cullin 3 (CUL3). All mutations result in up-regulation of the WNK signalling pathway which activates thiazide-sensitive Na-Cl cotransporters (NCC) in renal distal tubules, leading to salt retention and hypertension in PHAII. The mechanism underlying hypercalciuria in PHAII is unknown. To better understand the mechanisms leading to osteopenia in PHAII, we used KLHL3R528H/+ knock-in mice carrying the same mutation as some PHAII patients. As expected, KLHL3R528H/+ mutants exhibited hyperkalemia, hypernatremia, renal calcium wasting and increased phosphorylation of NCC in the kidney. Furthermore, KLHL3R528H/+ mutants showed elevated serum parathyroid hormone (PTH), increased bone resorption as demonstrated by elevated urinary collagen crosslinks excretion and increased osteoclast numbers in femoral cancellous bone, and reduced distal femoral cancellous bone BMD and volume as evidenced by pQCT and μCT analysis. Analysis of the expression of proteins involved in renal calcium transport revealed elevated membrane abundance of the fully glycosylated epithelial calcium channel TRPV5, decreased TRPV6 abundance, and unchanged calbindin D28k expression in KLHL3R528H/+ mutants. In contrast to the upregulated TRPV5 protein expression, TRPV5 phosphorylation was reduced in KLHL3R528H/+ mutants, suggesting downregulated TRPV5 activity. In line with a crosstalk between NCC activity and PTH-mediated TRPV5 activation, we found by 2-photon microscopy that the PTH-mediated increase in Ca2+ uptake in mouse distal tubular mpkDCT4 cells was enhanced by the NCC blocker chlorothiazide or by knockout of NCC. Taken together, our study provides a mechanistic explanation for the hypercalciuria and bone loss found in PHAII patients: elevated NCC activity in KLHL3R528H/+ mice blocks PTH-mediated TRPV5 activation, leading to renal PTH resistance with subsequent renal Ca wasting and a counter-regulatory PTH-induced bone loss

Influence of Grass Species and Sample Preparation on Ensiling Characteristics

Laboratory silos are considered a practical method of comparing a number of treatments (O\u27Kiely, 1993). Cherney et al. (2004) reported that vacuum-sealed polyethylene bags effectively ensiled corn silage samples in the laboratory. Grasses, with their inherently higher buffering capacities and lower sugar levels, generally are more difficult to ensile. Objectives were to evaluate the influence of species and chopping (whole vs. shredded) on pH and volatile fatty acid profile of grasses ensiled in vacuum-sealed polyethylene bags and to assess the suitability of this method as a laboratory ensiling method

Impact of Type II LRRK2 inhibitors on signaling and mitophagy

Much effort has been devoted to the development of selective inhibitors of the LRRK2 as a potential treatment for LRRK2 driven Parkinson's disease. In this study, we first compare the properties of Type I (GSK3357679A and MLi-2) and Type II (GZD-824, Rebastinib and Ponatinib) kinase inhibitors that bind to the closed or open conformations of the LRRK2 kinase domain, respectively. We show that Type I and Type II inhibitors suppress phosphorylation of Rab10 and Rab12, key physiological substrates of LRRK2 and also promote mitophagy, a process suppressed by LRRK2. Type II inhibitors also display higher potency towards wild-type LRRK2 compared with pathogenic mutants. Unexpectedly, we find that Type II inhibitors, in contrast with Type I compounds, fail to induce dephosphorylation of a set of well-studied LRRK2 biomarker phosphorylation sites at the N-terminal region of LRRK2, including Ser935. These findings emphasize that the biomarker phosphorylation sites on LRRK2 are likely reporting on the open vs closed conformation of LRRK2 kinase and that only inhibitors which stabilize the closed conformation induce dephosphorylation of these biomarker sites. Finally, we demonstrate that the LRRK2[A2016T] mutant which is resistant to MLi-2 Type 1 inhibitor, also induces resistance to GZD-824 and Rebastinib suggesting this mutation could be exploited to distinguish off target effects of Type II inhibitors. Our observations provide a framework of knowledge to aid with the development of more selective Type II LRRK2 inhibitors

Alessi 95 and the short period Cepheid SU Cassiopeiae

The parameters for the newly-discovered open cluster Alessi 95 are established on the basis of available photometric and spectroscopic data, in conjunction with new observations. Colour excesses for spectroscopically-observed B and A-type stars near SU Cas follow a reddening relation described by E(U-B)/E(B-V)=0.83+0.02*E(B-V), implying a value of R=Av/E(B-V)~2.8 for the associated dust. Alessi 95 has a mean reddening of E(B-V)_(B0)=0.35+-0.02 s.e., an intrinsic distance modulus of Vo-Mv=8.16+-0.04 s.e. (+-0.21 s.d.), d=429+-8 pc, and an estimated age of 10^8.2 yr from ZAMS fitting of available UBV, CCD BV, NOMAD, and 2MASS JHKs observations of cluster stars. SU Cas is a likely cluster member, with an inferred space reddening of E(B-V)=0.33+-0.02 and a luminosity of =-3.15+-0.07 s.e., consistent with overtone pulsation (P_FM=2.75 d), as also implied by the Cepheid's light curve parameters, rate of period increase, and Hipparcos parallaxes for cluster stars. There is excellent agreement of the distance estimates for SU Cas inferred from cluster ZAMS fitting, its pulsation parallax derived from the infrared surface brightness technique, and Hipparcos parallaxes, which all agree to within a few percent.Comment: Accepted for Publication (MNRAS

Lesões intestinais provocadas pela obstrução experimental do cólon menor equino com isquemia mural

O artigo não apresenta resumo

Modelo experimental de neurorafia em equinos

O artigo não apresenta resumo

A Motivating Exploration on Lunar Craters and Low-Energy Dynamics in the Earth -- Moon System

It is known that most of the craters on the surface of the Moon were created by the collision of minor bodies of the Solar System. Main Belt Asteroids, which can approach the terrestrial planets as a consequence of different types of resonance, are actually the main responsible for this phenomenon. Our aim is to investigate the impact distributions on the lunar surface that low-energy dynamics can provide. As a first approximation, we exploit the hyberbolic invariant manifolds associated with the central invariant manifold around the equilibrium point L_2 of the Earth - Moon system within the framework of the Circular Restricted Three - Body Problem. Taking transit trajectories at several energy levels, we look for orbits intersecting the surface of the Moon and we attempt to define a relationship between longitude and latitude of arrival and lunar craters density. Then, we add the gravitational effect of the Sun by considering the Bicircular Restricted Four - Body Problem. As further exploration, we assume an uniform density of impact on the lunar surface, looking for the regions in the Earth - Moon neighbourhood these colliding trajectories have to come from. It turns out that low-energy ejecta originated from high-energy impacts are also responsible of the phenomenon we are considering.Comment: The paper is being published in Celestial Mechanics and Dynamical Astronomy, vol. 107 (2010

Analysis of the LKB1-STRAD-MO25 complex

Mutations in the LKB1 tumour suppressor threonine kinase cause the inherited Peutz-Jeghers cancer syndrome and are also observed in some sporadic cancers. Recent work indicates that LKB1 exerts effects on metabolism, polarity and proliferation by phosphorylating and activating protein kinases belonging to the AMPK subfamily. In vivo, LKB1 forms a complex with STRAD, an inactive pseudokinase, and MO25, an armadillo repeat scaffolding-like protein. Binding of LKB1 to STRAD-MO25 activates LKB1 and re-localises it from the nucleus to the cytoplasm. To learn more about the inherent properties of the LKB1-STRAD-MO25 complex, we first investigated the activity of 34 point mutants of LKB1 found in human cancers and their ability to interact with STRAD and MO25. Interestingly, 12 of these mutants failed to interact with STRAD-MO25. Performing mutagenesis analysis, we defined two binding sites located on opposite surfaces of MO25α, which are required for the assembly of MO25α into a complex with STRADα and LKB1. In addition, we demonstrate that LKB1 does not require phosphorylation of its own T-loop to be activated by STRADα-MO25α, and discuss the possibility that this unusual mechanism of regulation arises from LKB1 functioning as an upstream kinase. Finally, we establish that STRADα, despite being catalytically inactive, is still capable of binding ATP with high affinity, but that this is not required for activation of LKB1. Taken together, our findings reinforce the functional importance of the binding of LKB1 to STRAD, and provide a greater understanding of the mechanism by which LKB1 is regulated and activated through its interaction with STRAD and MO25