TRPV6 Determines the Effect of Vitamin D3 on Prostate Cancer Cell Growth

Despite remarkable advances in the therapy and prevention of prostate cancer it is still the second cause of death from cancer in industrialized countries. Many therapies initially shown to be beneficial for the patients were abandoned due to the high drug resistance and the evolution rate of the tumors. One of the prospective therapeutical agents even used in the first stage clinical trials, 1,25-dihydroxyvitamin D3, was shown to be either unpredictable or inefficient in many cases. We have already shown that TRPV6 calcium channel, which is the direct target of 1,25-dihydroxyvitamin D3 receptor, positively controls prostate cancer proliferation and apoptosis resistance (Lehen'kyi et al., Oncogene, 2007). However, how the known 1,25-dihydroxyvitamin D3 antiproliferative effects may be compatible with the upregulation of pro-oncogenic TRPV6 channel remains a mystery. Here we demonstrate that in low steroid conditions 1,25-dihydroxyvitamin D3 upregulates the expression of TRPV6, enchances the proliferation by increasing the number of cells entering into S-phase. We show that these pro-proliferative effects of 1,25-dihydroxyvitamin D3 are directly mediated via the overexpression of TRPV6 channel which increases calcium uptake into LNCaP cells. The apoptosis resistance of androgen-dependent LNCaP cells conferred by TRPV6 channel is drastically inversed when 1,25-dihydroxyvitamin D3 effects were combined with the successful TRPV6 knockdown. In addition, the use of androgen-deficient DU-145 and androgen-insensitive LNCaP C4-2 cell lines allowed to suggest that the ability of 1,25-dihydroxyvitamin D3 to induce the expression of TRPV6 channel is a crucial determinant of the success or failure of 1,25-dihydroxyvitamin D3-based therapies

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Immobilization of lipid substrates: application on phospholipase A2 determination

The purpose of the study was to assess a fluorimetric assay for the determination of total phospholipase A2 (PLA2) activity in biological samples introducing the innovation of immobilized substrates on crosslinked polymeric membranes. The immobilized C12-NBD-PtdCho, a fluorescent analogue of phosphatidylcholine, exhibited excellent stability for 3 months at 4 °C and was not desorbed in the aqueous reaction mixture during analysis. The limit of detection was 0.5 pmol FA (0.2 pg) and the linear part of the response curve extended from 1 up to 190 nmol FA/h/mL sample. The intra- and inter-day relative standard deviations (%RSD), were ≤6 and ≤9 %, respectively. Statistical comparison with other fluorescent methods showed excellent correlation and agreement. Semiempirical calculations showed a fair amount of electrostatic interaction between the NBD-labeled substrate and the crosslinked polyvinyl alcohol with the styryl pyridinium residues (PVA-SbQ) material, from the plane of which, the sn-2 acyl chain of the phospholipid stands out and is accessible by PLA2. Atomic Force Microscopy revealed morphological alterations of the immobilized substrate after the reaction with PLA2. Mass spectrometry showed that only C12-NBD-FA, the PLA2 hydrolysis product, was detected in the reaction mixture, indicating that PLA2 recognizes PVA-SbQ/C12-NBD-PtdCho as a surface to perform catalysis.</p

Assessing and managing intensification in smallholder dairy systems for food and nutrition security in Sub-Saharan Africa

Smallholder farmers play an important part in the dairy value chain in Sub-Saharan Africa. Three technological approaches have been used to improve productivity. These are through, applying agricultural ecological processes (ecological intensification), utilising modern livestock breeding (genetic intensification), and socio-economic intensification. Ecological intensification includes continuous housing of cows applying a cut-and-carry feeding system, introduction of purpose-bred forages and pastures, and the introduction of agro-forestry within the dairy systems. Genetic intensification strategies include: importation of dairy breeds such as Holstein–Friesian (HF) and cross-breeding of the indigenous breeds with HF. Training and capacity-building activities to create sustainable livelihoods have been initiated for farming and technological practices of animal husbandry, but also to enhance appropriate leadership and corporative-building skills that would create and support an enabling environment for sustainability. These improvements and initiatives in the service delivery have been championed by national governments, development partner institutions, or non-governmental organisations through different programmes. Challenges of intensification include matching management to genetic potential of imported and cross-bred improved dairy breeds, ensuring low post-harvest losses, proper utilisation, and reducing environmental impact. Using examples from Kenya, Malawi, Mozambique, Tanzania, and Zambia, this paper reviews the management and assessment approaches used in fostering smallholder dairy development strategies and dairy’s contribution to sustainable livelihoods in the face of intensification