European Lung Cancer Working Party Clinical Practice Guidelines. Non-Small Cell Lung Cancer: III. Metastatic disease

The present guidelines on the management of advanced non-small cell lung cancer (NS CLC) were formulated by the ELCWP in October 2006. They are designed to answer the following twelve questions: 1) What benefits can be expected from chemotherapy and what are the treatment objectives? 2) What are the active chemotherapeutic drugs for which efficacy has been shown? 3) Which are the most effective platinum-based regimens? 4) Which is the indicated dosage of cisplatin? 5) Can carboplatin be substituted for cisplatin? 6) Which is the optimal number of cycles to be administered? 7) Can non-platinum based regimens be substituted for platinum based chemotherapy as first-line treatment? 8) Is there an indication for sequential chemotherapy? 9) What is the efficacy of salvage chemotherapy and which drugs should be used in that indication? 10) What is the place of targeted therapies? 11) What is the place of chemotherapy in the management of a patient with brain metastases? 12) Which specific drugs can be used for the patient with bone metastases

Genome-wide association for milk production and lactation curve parameters in Holstein dairy cows

The aim of this study was to identify genomic regions associated with 305-day milk yield and lactation curve parameters on primiparous (n = 9,910) and multiparous (n = 11,158) Holstein cows. The SNP solutions were estimated using a weighted single-step genomic BLUP approach and imputed high-density panel (777k) genotypes. The proportion of genetic variance explained by windows of 50 consecutive SNP (with an average of 165 Kb) was calculated, and regions that accounted for more than 0.50% of the variance were used to search for candidate genes. Estimated heritabilities were 0.37, 0.34, 0.17, 0.12, 0.30 and 0.19, respectively, for 305-day milk yield, peak yield, peak time, ramp, scale and decay for primiparous cows. Genetic correlations of 305-day milk yield with peak yield, peak time, ramp, scale and decay in primiparous cows were 0.99, 0.63, 0.20, 0.97 and -0.52, respectively. The results identified three windows on BTA14 associated with 305-day milk yield and the parameters of lactation curve in primi- and multiparous cows. Previously proposed candidate genes for milk yield supported by this work include GRINA, CYHR1, FOXH1, TONSL, PPP1R16A, ARHGAP39, MAF1, OPLAH and MROH1, whereas newly identified candidate genes are MIR2308, ZNF7, ZNF34, SLURP1, MAFA and KIFC2 (BTA14). The protein lipidation biological process term, which plays a key role in controlling protein localization and function, was identified as the most important term enriched by the identified genes

Expert-based development of a generic HACCP-based risk management system to prevent critical negative energy balance in dairy herds

The objective of this study was to develop a generic risk management system based on the Hazard Analysis and Critical Control Point (HACCP) principles for the prevention of critical negative energy balance (NEB) in dairy herds using an expert panel approach. In addition, we discuss the advantages and limitations of the system in terms of implementation in the individual dairy herd. For the expert panel, we invited 30 researchers and advisors with expertise in the field of dairy cow feeding and/or health management from eight European regions. They were invited to a Delphi-based set-up that included three inter-correlated questionnaires in which they were asked to suggest risk factors for critical NEB and to score these based on 'effect' and 'probability'. Finally, the experts were asked to suggest critical control points (CCPs) specified by alarm values, monitoring frequency and corrective actions related to the most relevant risk factors in an operational farm setting. A total of 12 experts (40 %) completed all three questionnaires. Of these 12 experts, seven were researchers and five were advisors and in total they represented seven out of the eight European regions addressed in the questionnaire study. When asking for suggestions on risk factors and CCPs, these were formulated as 'open questions', and the experts' suggestions were numerous and overlapping. The suggestions were merged via a process of linguistic editing in order to eliminate doublets. The editing process revealed that the experts provided a total of 34 CCPs for the 11 risk factors they scored as most important. The consensus among experts was relatively high when scoring the most important risk factors, while there were more diverse suggestions of CCPs with specification of alarm values and corrective actions. We therefore concluded that the expert panel approach only partly succeeded in developing a generic HACCP for critical NEB in dairy cows. We recommend that the output of this paper is used to inform key areas for implementation on the individual dairy farm by local farm teams including farmers and their advisors, who together can conduct herd-specific risk factor profiling, organise the ongoing monitoring of herd-specific CCPs, as well as implement corrective actions when CCP alarm values are exceeded

Yeast Gdt1 is a Golgi-localized calcium transporter required for stress-induced calcium signaling and protein glycosylation

Calcium signaling depends on a tightly regulated set of pumps, exchangers, and channels that are responsible for controlling calcium fluxes between the different subcellular compartments of the eukaryotic cell. It have been recently reported in our lab that two members of the highly-conserved UPF0016 family, human TMEM165 and budding yeast Gdt1p, are functionally related and form a new group of Golgi-localized cation/Ca2+ exchangers. Both share topological characteristics reminiscent of the cation/calcium exchanger superfamily. They are composed of two hydrophobic regions containing a highly conserved motifs separated by a large cytosolic loop. Defects in the human protein TMEM165 are known to cause a subtype of Congenital Disorders of Glycosylation. The Ca2+ transport activity of Gdt1p was demonstrated using an assay based on the heterologous expression of GDT1 in the bacterium Lactococcus lactis. The Ca2+ uptake activity in cell expressing GDT1 was dependent on the external pH, indicating that Gdt1p may act as a Ca2+/H+ antiporter. This work focuses on the functional and structural study of Gdt1p in the yeast Saccharomyces cerevisiae. We firstly found that Gdt1p controls cellular calcium content and plays a major role in the calcium response following an osmotic shock when the Golgi calcium pump, Pmr1p, is absent. Importantly, we also discovered that, in the presence of a high concentration of external Ca2+, Gdt1p is required for glycosylation of carboxypeptidase Y and the glucanosyltransferase Gas1p. Additionally, we highlighted that Gdt1p levels are controlled by external Ca2+ via a mechanism involving the calcineurin signaling pathway along with vacuolar proteolysis. Finally, by site-directed mutagenesis and characterization of the mutants, we found that the cations binding pocket of Gdt1p is most likely composed of glutamate, aspartate, serine and threonine residues located in the repeated motifs.(AGRO - Sciences agronomiques et ingénierie biologique) -- UCL, 201

Molecular evolution of a novel family of putative calcium transporters.

The UPF0016 family is a group of uncharacterized membrane proteins, well conserved through evolution and defined by the presence of one or two copies of an E-Φ-G-D-(KR)-(ST) consensus motif. Our previous results have shown that two members of this family, the human TMEM165 and the budding yeast Gdt1p, are functionally related and might form a new group of cation/Ca2+ exchangers. Most members of the family are made of two homologous clusters of three transmembrane spans, separated by a central loop and assembled with an opposite orientation in the membrane. However, some bacterial members of the family have only one cluster of transmembrane domains. Among these 'single-domain membrane proteins' some cyanobacterial members were found as pairs of adjacent genes within the genome, but each gene was slightly different. We performed a bioinformatic analysis to propose the molecular evolution of the UPF0016 family and the emergence of the antiparallel topology. Our hypotheses were confirmed experimentally using functional complementation in yeast. This suggests an important and conserved function for UPF0016 proteins in a fundamental cellular process. We also show that members of the UPF0016 family share striking similarities, but no primary sequence homology, with members of the cation/Ca2+ exchangers (CaCA) superfamily. Such similarities could be an example of convergent evolution, supporting the previous hypothesis that members of the UPF0016 family are cation/Ca2+ exchangers

A new pH sensor localized in the Golgi apparatus of Saccharomyces cerevisiae reveals unexpected roles of Vph1p and Stv1p isoforms.

The gradual acidification of the secretory pathway is conserved and extremely important for eukaryotic cells, but until now there was no pH sensor available to monitor the pH of the early Golgi apparatus in Saccharomyces cerevisiae. Therefore, we developed a pHluorin-based sensor for in vivo measurements in the lumen of the Golgi. By using this new tool we show that the cis- and medial-Golgi pH is equal to 6.6-6.7 in wild type cells during exponential phase. As expected, V-ATPase inactivation results in a near neutral Golgi pH. We also uncover that surprisingly Vph1p isoform of the V-ATPase is prevalent to Stv1p for Golgi acidification. Additionally, we observe that during changes of the cytosolic pH, the Golgi pH is kept relatively stable, mainly thanks to the V-ATPase. Eventually, this new probe will allow to better understand the mechanisms involved in the acidification and the pH control within the secretory pathway

The yeast Gdt1 protein mediates the exchange of H+ for Ca2+ and Mn2+ influencing the Golgi pH

The GDT1 family is broadly spread and highly conserved among living organisms. GDT1 members have functions in key processes like glycosylation in humans and yeasts and photo-synthesis in plants. These functions are mediated by their ability to transport ions. While transport of Ca2+ or Mn2+ is well established for several GDT1 members, their transport mechanism is poorly understood. Here, we demonstrate that H + ions are transported in exchange for Ca 2+ and Mn 2+ cations by the Golgi-localized yeast Gdt1 protein. We performed direct transport measurement across a biological membrane by expressing Gdt1p in Lactococcus lactis bacterial cells and by recording either the extracellular pH or the intracellular pH during the application of Ca2+ , Mn2+ or H + gradients. Besides, in vivo cytosolic and Golgi pH measurements were performed in Saccharomyces cerevisiae with genetically encoded pH probes targeted to those subcellular compartments. These data point out that the flow of H + ions carried by Gdt1p could be reversed according to the physiological conditions. Together, our experiments unravel the influence of the relative concentration gradients for Gdt1p-mediated H + transport and pave the way to decipher the regulatory mechanisms driving the activity of GDT1 orthologs in various biological contexts

K+-specific importers Trk1 and Trk2 play different roles in Ca2+ homeostasis and signalling in Saccharomyces cerevisiae cells.

The maintenance of K+ and Ca2+ homeostasis is crucial for many cellular functions. Potassium is accumulated in cells at high concentrations, while the cytosolic level of calcium, to ensure its signalling function, is kept at low levels and transiently increases in response to stresses. We examined Ca2+ homeostasis and Ca2+ signalling in Saccharomyces cerevisiae strains lacking plasma-membrane K+ influx (Trk1 and Trk2) or efflux (Tok1, Nha1 and Ena1-5) systems. The lack of K+ exporters slightly increased the cytosolic Ca2+, but did not alter the Ca2+ tolerance or Ca2+-stress response. In contrast, the K+-importers Trk1 and Trk2 play important and distinct roles in the maintenance of Ca2+ homeostasis. The presence of Trk1 was vital mainly for the growth of cells in the presence of high extracellular Ca2+, whilst the lack of Trk2 doubled steady-state intracellular Ca2+ levels. The absence of both K+ importers highly increased the Ca2+ response to osmotic or CaCl2 stresses and altered the balance between Ca2+ flux from external media and intracellular compartments. In addition, we found Trk2 to be important for the tolerance to high KCl and hygromycin B in cells growing on minimal media. All the data describe new interconnections between potassium and calcium homeostasis in S. cerevisiae

Acidic and uncharged polar residues in the consensus motifs of the yeast Ca2+ transporter Gdt1p are required for calcium transport

The UPF0016 family is a recently identified group of poorly characterized membrane proteins whose function is conserved through evolution and that are defined by the presence of one or two copies of the E-φ-G-D-[KR]-[TS] consensus motif in their transmembrane domain. We previously showed that two members of this family, the human TMEM165 and the budding yeast Gdt1p, are functionally related and are likely to form a new group of Ca2+ transporters. Mutations in TMEM165 have been demonstrated to cause a new type of rare human genetic diseases denominated as Congenital Disorders of Glycosylation. Using site-directed mutagenesis, we generated seventeen mutations in the yeast Golgi-localized Ca2+ transporter Gdt1p. Single alanine substitutions were targeted to the highly conserved consensus motifs, four acidic residues localized in the central cytosolic loop, and the arginine at position 71. The mutants were screened in a yeast strain devoid of both the endogenous Gdt1p exchanger and Pmr1p, the Ca2+-ATPase of the Golgi apparatus. We show here that acidic and polar uncharged residues of the consensus motifs play a crucial role in calcium tolerance and calcium transport activity, and are therefore likely to be architectural components of the cation binding site of Gdt1p. Importantly, we confirm the essential role of the E53 residue whose mutation in humans triggers Congenital Disorders of Glycosylation

Gdt1p, a new player involved in calcium homeostasis in Saccharomyces cerevisiae? Development of organelles-localized calcium and pH-sensitive probes.

The Ca2+ ion is a ubiquitous intracellular messenger involved in various signaling processes. The basal concentration of cytosolic calcium is maintained at a very low level. Upon stimulation, a transient increase of cytosolic Ca2+ originated from internal and/or external stores is observed. Therefore, a complex toolkit of pumps, exchangers and channels localized in the different compartments of the cell are essential to tightly control this mechanism. Although a significant number of those are already well known, there are still some lacking actors in the Saccharomyces cerevisiae Ca2+-signaling toolkit. Recently, we have identified a novel family of putative Ca2+/H+ antiporters (UPF0016), whose Gdt1p is the yeast member. Gdt1p is a six-transmembrane spans protein located to the Golgi apparatus. It possesses a characteristic conserved hydrophilic motif in two transmembrane spans. Phenotypical assays and cytosolic Ca2+ concentration measurement suggested that Gdt1p could play an important role in Ca2+ homeostasis. However, the precise function of Gdt1p remains unclear. In order to elucidate the physiological function of Gdt1p related to calcium and pH homeostasis within the secretory pathway, we try to get direct evidences of Ca2+/H+ transport. For this purpose, we are addressing genetically-encoded Ca2+ sensors and H+ probes to the lumen of different organelles (endoplasmic reticulum, Golgi apparatus, vacuole). Aequorin, GAP (GFP-Aequorin Probe) and pHluorin are bioluminescent/fluorescent probes useful for Ca2+ or pH measurement, respectively. So far, we correctly localized these sensors to the endoplasmic reticulum and the Golgi apparatus lumens. However, the low expression level does not allow us to distinguish specific signal from noise. The next challenge is then to increase the expression level of these probes while preserving the right localization