Final report : SIM comparison in mass standards SIM.M.M-K5

This report summarizes the results of a SIM comparison in masss carried out between 7 NMIs. Five mass standards with nominal values 2 kg, 200 g, 50 g, 1 g and 200 mg have been circulated by the NMIs. The results reported by the participants are consistent with each other and with the key comparison reference valu of the comparison CCM.M-K5 to which the present comparison has been linked.Fil: Becerra, L. O. Centro Nacional de Metrología (CENAM); MéxicoFil: Peña, L. M. Centro Nacional de Metrología (CENAM); MéxicoFil: Luján, L. Centro Nacional de Metrología (CENAM); MéxicoFil: Díaz, J. C. Centro Nacional de Metrología (CENAM); MéxicoFil: Centeno, , L. M. Centro Nacional de Metrología (CENAM); MéxicoFil: Loayza, V. M. Instituto Nacional de Metrologia, Qualidade e Tecnologia (INMetro); BrasilFil: Cacais, F. A. Instituto Nacional de Metrologia, Qualidade e Tecnologia (INMetro); BrasilFil: Ramos, O. Laboratorio Costarricense de Metrología (LaCoMet); Costa RicaFil: Rodriguez, S. Laboratorio Costarricense de Metrología (LaCoMet); Costa RicaFil: Garcia, F. Centro de Estudios, Medición y Certificación de Calidad (CESMEC); ChileFil: Leyton, F. Centro de Estudios, Medición y Certificación de Calidad (CESMEC); ChileFil: Santo, C. Laboratorio Tecnológico del Uruguay (LATU); UruguayFil: Caceres, J. Laboratorio Tecnológico del Uruguay (LATU); UruguayFil: Kornblit, F. Instituto Nacional de Tecnología Industrial (INTI); ArgentinaFil: Leiblich, J. Instituto Nacional de Tecnología Industrial (INTI); ArgentinaFil: Claude, J. National Research Council (NRC); Canad

Final report : SIM comparison in mass standards SIM.M.M-K4

This report summarizes the results of a SIM comparison in masss carried out between 7 NMIs. Five mass standards with nominal values 2 kg, 200 g, 50 g, 1 g and 200 mg have been circulated by the NMIs. The results reported by the participants are consistent with each other and with the key comparison reference valu of the comparison CCM.M-K5 to which the present comparison has been linked.Fil: Becerra, L. O. Centro Nacional de Metrología (CENAM); MéxicoFil: Peña, L. M. Centro Nacional de Metrología (CENAM); MéxicoFil: Luján, L. Centro Nacional de Metrología (CENAM); MéxicoFil: Díaz, J. C. Centro Nacional de Metrología (CENAM); MéxicoFil: Centeno, , L. M. Centro Nacional de Metrología (CENAM); MéxicoFil: Loayza, V. M. Instituto Nacional de Metrologia, Qualidade e Tecnologia (INMetro); BrasilFil: Cacais, F. A. Instituto Nacional de Metrologia, Qualidade e Tecnologia (INMetro); BrasilFil: Ramos, O. Laboratorio Costarricense de Metrología (LaCoMet); Costa RicaFil: Rodriguez, S. Laboratorio Costarricense de Metrología (LaCoMet); Costa RicaFil: Garcia, F. Centro de Estudios, Medición y Certificación de Calidad (CESMEC); ChileFil: Leyton, F. Centro de Estudios, Medición y Certificación de Calidad (CESMEC); ChileFil: Santo, C. Laboratorio Tecnológico del Uruguay (LATU); UruguayFil: Caceres, J. Laboratorio Tecnológico del Uruguay (LATU); UruguayFil: Kornblit, F. Instituto Nacional de Tecnología Industrial (INTI); ArgentinaFil: Leiblich, J. Instituto Nacional de Tecnología Industrial (INTI); ArgentinaFil: Claude, J. National Research Council (NRC); Canad

Regulation of Dense-Core Granule Replenishment by Autocrine BMP Signalling in Drosophila Secondary Cells

Regulated secretion by glands and neurons involves release of signalling molecules and enzymes selectively concentrated in dense-core granules (DCGs). Although we understand how many secretagogues stimulate DCG release, how DCG biogenesis is then accelerated to replenish the DCG pool remains poorly characterised. Here we demonstrate that each prostate-like secondary cell (SC) in the paired adult Drosophila melanogaster male accessory glands contains approximately ten large DCGs, which are loaded with the Bone Morphogenetic Protein (BMP) ligand Decapentaplegic (Dpp). These DCGs can be marked in living tissue by a glycophosphatidylinositol (GPI) lipid-anchored form of GFP. In virgin males, BMP signalling is sporadically activated by constitutive DCG secretion. Upon mating, approximately four DCGs are typically released immediately, increasing BMP signalling, primarily via an autocrine mechanism. Using inducible knockdown specifically in adult SCs, we show that secretion requires the Soluble NSF Attachment Protein, SNAP24. Furthermore, mating-dependent BMP signalling not only promotes cell growth, but is also necessary to accelerate biogenesis of new DCGs, restoring DCG number within 24 h. Our analysis therefore reveals an autocrine BMP-mediated feedback mechanism for matching DCG release to replenishment as secretion rates fluctuate, and might explain why in other disease-relevant systems, like pancreatic β-cells, BMP signalling is also implicated in the control of secretion

Microguards and micromessengers of the genome

The regulation of gene expression is of fundamental importance to maintain organismal function and integrity and requires a multifaceted and highly ordered sequence of events. The cyclic nature of gene expression is known as ‘transcription dynamics’. Disruption or perturbation of these dynamics can result in significant fitness costs arising from genome instability, accelerated ageing and disease. We review recent research that supports the idea that an important new role for small RNAs, particularly microRNAs (miRNAs), is in protecting the genome against short-term transcriptional fluctuations, in a process we term ‘microguarding’. An additional emerging role for miRNAs is as ‘micromessengers’—through alteration of gene expression in target cells to which they are trafficked within microvesicles. We describe the scant but emerging evidence that miRNAs can be moved between different cells, individuals and even species, to exert biologically significant responses. With these two new roles, miRNAs have the potential to protect against deleterious gene expression variation from perturbation and to themselves perturb the expression of genes in target cells. These interactions between cells will frequently be subject to conflicts of interest when they occur between unrelated cells that lack a coincidence of fitness interests. Hence, there is the potential for miRNAs to represent both a means to resolve conflicts of interest, as well as instigate them. We conclude by exploring this conflict hypothesis, by describing some of the initial evidence consistent with it and proposing new ideas for future research into this exciting topic

Lactate-Dehydrogenase 5 is overexpressed in non-small cell lung cancer and correlates with the expression of the transketolase-like protein 1

<p>Abstract</p> <p>Aims</p> <p>As one of the five Lactate dehydrogenase (LDH) isoenzymes, LDH5 has the highest efficiency to catalyze pyruvate transformation to lactate. LDH5 overexpression in cancer cells induces an upregulated glycolytic metabolism and reduced dependence on the presence of oxygen. Here we analyzed LDH5 protein expression in a well characterized large cohort of primary lung cancers in correlation to clinico-pathological data and its possible impact on patient survival.</p> <p>Methods</p> <p>Primary lung cancers (n = 269) and non neoplastic lung tissue (n = 35) were tested for LDH5 expression by immunohistochemistry using a polyclonal LDH5 antibody (ab53010). The results of LDH5 expression were correlated to clinico-pathological data as well as to patient's survival. In addition, the results of the previously tested Transketolase like 1 protein (TKTL1) expression were correlated to LDH5 expression.</p> <p>Results</p> <p>89.5% (n = 238) of NSCLC revealed LDH5 expression whereas LDH5 expression was not detected in non neoplastic lung tissues (n = 34) (p < 0.0001). LDH5 overexpression was associated with histological type (adenocarcinoma = 57%, squamous cell carcinoma = 45%, large cell carcinoma = 46%, p = 0.006). No significant correlation could be detected with regard to TNM-stage, grading or survival. A two sided correlation between the expression of TKTL1 and LDH5 could be shown (p = 0.002) within the overall cohort as well as for each grading and pN group. A significant correlation between LDH5 and TKTL1 within each histologic tumortype could not be revealed.</p> <p>Conclusions</p> <p>LDH5 is overexpressed in NSCLC and could hence serve as an additional marker for malignancy. Furthermore, LDH5 correlates positively with the prognostic marker TKTL1. Our results confirm a close link between the two metabolic enzymes and indicate an alteration in the glucose metabolism in the process of malignant transformation.</p

Lactate Dehydrogenase-B Is Silenced by Promoter Methylation in a High Frequency of Human Breast Cancers

Objective: Under normoxia, non-malignant cells rely on oxidative phosphorylation for their ATP production, whereas cancer cells rely on Glycolysis; a phenomenon known as the Warburg effect. We aimed to elucidate the mechanisms contributing to the Warburg effect in human breast cancer. Experimental design: Lactate Dehydrogenase (LDH) isoenzymes were profiled using zymography. LDH-B subunit expression was assessed by reverse transcription PCR in cells, and by Immunohistochemistry in breast tissues. LDH-B promoter methylation was assessed by sequencing bisulfite modified DNA. Results: Absent or decreased expression of LDH isoenzymes 1-4, were seen in T-47D and MCF7 cells. Absence of LDH-B mRNA was seen in T-47D cells, and its expression was restored following treatment with the demethylating agent 5'Azacytadine. LDH-B promoter methylation was identified in T-47D and MCF7 cells, and in 25/ 25 cases of breast cancer tissues, but not in 5/ 5 cases of normal breast tissues. Absent immuno-expression of LDH-B protein (<10% cells stained), was seen in 23/ 26 (88%) breast cancer cases, and in 4/8 cases of adjacent ductal carcinoma in situ lesions. Exposure of breast cancer cells to hypoxia (1% O2), for 48 hours resulted in significant increases in lactate levels in both MCF7 (14.0 fold, p = 0.002), and T-47D cells (2.9 fold, p = 0.009), but not in MDA-MB-436 (-0.9 fold, p = 0.229), or MCF10AT (1.2 fold, p = 0.09) cells. Conclusions: Loss of LDH-B expression is an early and frequent event in human breast cancer occurring due to promoter methylation, and is likely to contribute to an enhanced glycolysis of cancer cells under hypoxia

Expression and Functional Studies of Ubiquitin C-Terminal Hydrolase L1 Regulated Genes

Deubiquitinating enzymes (DUBs) have been increasingly implicated in regulation of cellular processes, but a functional role for Ubiquitin C-terminal Hydrolases (UCHs), which has been largely relegated to processing of small ubiquitinated peptides, remains unexplored. One member of the UCH family, UCH L1, is expressed in a number of malignancies suggesting that this DUB might be involved in oncogenic processes, and increased expression and activity of UCH L1 have been detected in EBV-immortalized cell lines. Here we present an analysis of genes regulated by UCH L1 shown by microarray profiles obtained from cells in which expression of the gene was inhibited by RNAi. Microarray data were verified with subsequent real-time PCR analysis. We found that inhibition of UCH L1 activates genes that control apoptosis, cell cycle arrest and at the same time suppresses expression of genes involved in proliferation and migration pathways. These findings are complemented by biological assays for apoptosis, cell cycle progression and migration that support the data obtained from microarray analysis, and suggest that the multi-functional molecule UCH L1 plays a role in regulating principal pathways involved in oncogenesis

Evolutionary Rate Covariation Identifies New Members of a Protein Network Required for Drosophila melanogaster Female Post-Mating Responses

Seminal fluid proteins transferred from males to females during copulation are required for full fertility and can exert dramatic effects on female physiology and behavior. In Drosophila melanogaster, the seminal protein sex peptide (SP) affects mated females by increasing egg production and decreasing receptivity to courtship. These behavioral changes persist for several days because SP binds to sperm that are stored in the female. SP is then gradually released, allowing it to interact with its female-expressed receptor. The binding of SP to sperm requires five additional seminal proteins, which act together in a network. Hundreds of uncharacterized male and female proteins have been identified in this species, but individually screening each protein for network function would present a logistical challenge. To prioritize the screening of these proteins for involvement in the SP network, we used a comparative genomic method to identify candidate proteins whose evolutionary rates across the Drosophila phylogeny co-vary with those of the SP network proteins. Subsequent functional testing of 18 co-varying candidates by RNA interference identified three male seminal proteins and three female reproductive tract proteins that are each required for the long-term persistence of SP responses in females. Molecular genetic analysis showed the three new male proteins are required for the transfer of other network proteins to females and for SP to become bound to sperm that are stored in mated females. The three female proteins, in contrast, act downstream of SP binding and sperm storage. These findings expand the number of seminal proteins required for SP's actions in the female and show that multiple female proteins are necessary for the SP response. Furthermore, our functional analyses demonstrate that evolutionary rate covariation is a valuable predictive tool for identifying candidate members of interacting protein networks. © 2014 Findlay et al

Active surveillance of small renal masses offers short-term oncological efficacy equivalent to radical and partial nephrectomy.

UNLABELLED: Study Type - Therapy (case series) Level of Evidence 4. What's known on the subject? and What does the study add? Active surveillance of small renal masses has traditionally been reserved for elderly patients deemed unfit for surgery or ablation. There is increasing evidence showing the safety of active surveillance in the management of small renal masses. In this retrospective study we compared outcomes for patients with small renal masses managed with active surveillance, radical nephrectomy and partial nephrectomy. We showed that active surveillance was safe and appeared as effective as immediate surgery in the management of small renal tumours. OBJECTIVE: • To compare the oncological outcomes of active surveillance (AS), radical nephrectomy (RN) and partial nephrectomy (PN) in the management of T1a small renal masses (SRMs). PATIENTS AND METHODS: • At present AS is used in the treatment of SRMs in elderly patients with multiple co-morbidities or in those who decline surgery. • We identified all patients with T1a SRMs managed with RN, PN or AS. • Retrospective data were collected from patient case records with survival data and cause of death cross-referenced with the Oxford Cancer Intelligence Unit. RESULTS: • A total of 202 patients with 234 T1a SRMs (solid or Bosniak IV) were identified; 71 patients were managed with AS, 41 with an RN and 90 by PN. • Over a median follow-up of 34 months the mean growth rate on AS was 0.21 cm/year with 53% of SRMs managed with AS showing negative or zero growth. • No statistically significant difference was observed in overall (OS) and cancer-specific (CSS) survival for AS, RN and PN (AS-CSS 98.6%, AS-OS 83%; RN-CSS 92.6%, RN-OS 80.4%; PN-CSS 96.6%, PN-OS 90.0%). CONCLUSIONS: • Active surveillance of SRMs offers oncological efficacy equivalent to surgery in the short/intermediate term. • The results of this study support a multicentre prospective randomized controlled trial designed to compare the oncological efficacy of AS and surgery

Mating induces switch from hormone-dependent to hormone-independent steroid receptor–mediated growth in Drosophila secondary cells

Male reproductive glands like the mammalian prostate and the paired Drosophila melanogaster accessory glands secrete seminal fluid components that enhance fecundity. In humans, the prostate, stimulated by environmentally regulated endocrine and local androgens, grows throughout adult life. We previously showed that in fly accessory glands, secondary cells (SCs) and their nuclei also grow in adults, a process enhanced by mating and controlled by bone morphogenetic protein (BMP) signalling. Here, we demonstrate that BMP-mediated SC growth is dependent on the receptor for the developmental steroid ecdysone, whose concentration is reported to reflect sociosexual experience in adults. BMP signalling appears to regulate ecdysone receptor (EcR) levels via one or more mechanisms involving the EcR's N terminus or the RNA sequence that encodes it. Nuclear growth in virgin males is dependent on ecdysone, some of which is synthesised in SCs. However, mating induces additional BMP-mediated nuclear growth via a cell type-specific form of hormone-independent EcR signalling, which drives genome endoreplication in a subset of adult SCs. Switching to hormone-independent endoreplication after mating allows growth and secretion to be hyperactivated independently of ecdysone levels in SCs, permitting more rapid replenishment of the accessory gland luminal contents. Our data suggest mechanistic parallels between this physiological, behaviour-induced signalling switch and altered pathological signalling associated with prostate cancer progression