Proteomics and metabolomics in cancer drug development

In this review article, the main recent advancements in the field of proteomics and metabolomics and their application in cancer research are described. In the second part of the review the main metabolic alterations observed in cancer cells are thoroughly dissected, especially those involving anabolic pathways and NADPH-generating pathways, which indirectly affect anabolic reactions, other than the maintenance of the redox poise. Alterations to mitochondrial pathways and thereby deriving oncometabolites are also detailed. The third section of the review is a discussion of how and to what extent (mutations to) tumor suppressors and oncogenes end up influencing cancer cell metabolism and cell fate, either promoting survival and proliferation or autophagy and apoptosis. In the last section of the review, an overview is provided of therapeutic strategies that make use of metabolic reprogramming approaches.     

Emerging liquid chromatography–mass spectrometry technologies improving dried blood spot analysis

Dried blood spots (DBS), a micro blood sampling technique, has recently gained interest in drug discovery and development due to its inherent advantages over the conventional whole blood, plasma or serum sample collection. Since the regulatory authorities have agreed to the use of blood as an acceptable biological matrix for drug exposure measurements, its applications have been extended not only to therapeutic drug monitoring but also to toxicokinetic and pharmacokinetic studies. The pharmaceutical industry is keen to promote DBS as a prominent tool in bioanalytical applications due to the financial, ethical and organizational issues involved in clinical trials. This could be accomplished due to the latest advances in modern analytical technology, particularly liquid chromatography–mass spectrometry. The present review discusses some of the emerging liquid chromatography–mass spectrometry technologies in improving DBS analysis for its innovative applications in the development of new drugs.     

Conditional and domain‐specific inactivation of the Tsc2 gene in neural progenitor cells

Tuberous sclerosis complex (TSC) is a genetic disease characterized by multiorgan benign tumors as well as neurological manifestations. Epilepsy and autism are two of the more prevalent neurological complications and are usually severe. TSC is caused by mutations in either the TSC1 (encodes hamartin) or the TSC2 (encodes tuberin) genes with TSC2 mutations being associated with worse outcomes. Tuberin contains a highly conserved GTPase‐activating protein (GAP) domain that indirectly inhibits mammalian target of rapamycin complex 1 (mTORC1). mTORC1 dysregulation is currently thought to cause much of the pathogenesis in TSC but mTORC1‐independent mechanisms may also contribute. We generated a novel conditional allele of Tsc2 by flanking exons 36 and 37 with loxP sites. Mice homozygous for this knock‐in Tsc2 allele are viable and fertile with normal appearing growth and development. Exposure to Cre recombinase then creates an in‐frame deletion involving critical residues of the GAP domain. Homozygous conditional mutant mice generated using Emx1^Cre have increased cortical mTORC1 signaling, severe developmental brain anomalies, seizures, and die within 3 weeks. We found that the normal levels of the mutant Tsc2 mRNA, though GAP‐deficient tuberin protein, appear unstable and rapidly degraded. This novel animal model will allow further study of tuberin function including the requirement of the GAP domain for protein stability. genesis 51:284–292. © 2013 Wiley Periodicals, Inc.     

Response to ocean acidification in larvae of a large tropical marine fish,Rachycentron canadum

Currently, ocean acidification is occurring at a faster rate than at any time in the last 300 million years, posing an ecological challenge to marine organisms globally. There is a critical need to understand the effects of acidification on the vulnerable larval stages of marine fishes, as there is potential for large ecological and economic impacts on fish populations and the human economies that rely on them. We expand upon the narrow taxonomic scope found in the literature today, which overlooks many life history characteristics of harvested species, by reporting on the larvae of Rachycentron canadum (cobia), a large, highly mobile, pelagic‐spawning, widely distributed species with a life history and fishery value contrasting other species studied to date. We raised larval cobia through the first 3 weeks of ontogeny under conditions of predicted future ocean acidification to determine effects on somatic growth, development, otolith formation, swimming ability, and swimming activity. Cobia exhibited resistance to treatment effects on growth, development, swimming ability, and swimming activity at 800 and 2100 μatm pCO₂. However, these scenarios resulted in a significant increase in otolith size (up to 25% larger area) at the lowest pCO₂ levels reported to date, as well as the first report of significantly wider daily otolith growth increments. When raised under more extreme scenarios of 3500 and 5400 μatm pCO₂, cobia exhibited significantly reduced size‐at‐age (up to 25% smaller) and a 2–3 days developmental delay. The robust nature of cobia may be due to the naturally variable environmental conditions this species currently encounters throughout ontogeny in coastal environments, which may lead to an increased acclimatization ability even during long‐term exposure to stressors.     

Browning attenuates murine white adipose tissue expansion during postnatal development

During postnatal development of mice distinct white adipose tissue depots display a transient appearance of brown-like adipocytes. These brite (brown in white) adipocytes share characteristics with classical brown adipocytes including a multilocular appearance and the expression of the thermogenic protein uncoupling protein 1. In this study, we compared two inbred mouse strains 129S6sv/ev and C57BL6/N known for their different propensity to diet-induced obesity. We observed transient browning in retroperitoneal and inguinal adipose tissue depots of these two strains. From postnatal day 10 to 20 the increase in the abundance of multilocular adipocytes and uncoupling protein 1 expression was higher in 129S6sv/ev than in C57BL6/N pups. The parallel increase in the mass of the two fat depots was attenuated during this browning period. Conversely, epididymal white and interscapular brown adipose tissue displayed a steady increase in mass during the first 30 days of life. In this period, 129S6sv/ev mice developed a significantly higher total body fat mass than C57BL6/N. Thus, while on a local depot level a high number of brite cells is associated with the attenuation of adipose tissue expansion the strain comparison reveals no support for a systemic impact on energy balance. This article is part of a Special Issue entitled Brown and White Fat: From Signaling to Disease.     

A role for the miR396/GRF network in specification of organ type during flower development,as supported by ectopic expression of Populus trichocarpa miR396c in transgenic tobacco

The MIR396 family, composed of ath‐miR396a and ath‐miR396b in Arabidopsis, is conserved among plant species and is known to target the Growth‐Regulating Factor (GRF) gene family. ath‐miR396 overexpressors or grf mutants are characterised by small and narrow leaves and show embryogenic defects such as cotyledon fusion. Heterologous expression of ath‐miR396a has been reported in tobacco and resulted in reduction of the expression of three NtGRF genes. In this study, the precursor of the Populus trichocarpa ptc‐miR396c, with a mature sequence identical to ath‐miR396b, was expressed under control of the CaMV35S promoter in tobacco. Typical phenotypes of GRF down‐regulation were observed, including cotyledon fusion and lack of shoot apical meristem (SAM). At later stage of growth, transgenic plants had delayed development and altered specification of organ type during flower development. The third and fourth whorls of floral organs were modified into stigmatoid anthers and fasciated carpels, respectively. Several NtGRF genes containing a miR396 binding site were found to be down‐regulated, and the cleavage of their corresponding mRNA at the miR396 binding site was confirmed for two of them using RACE‐PCR analysis. The data obtained agree with the functional conservation of the miR396 family in plants and suggest a role for the miR396/GRF network in determination of floral organ specification.