Recombinant protein production by large-scale transient gene expression in mammalian cells : state of the art and future perspectives

The expansion of the biologics pipeline depends on the identification of candidate proteins for clinical trials. Speed is one of the critical issues, and the rapid production of high quality, research-grade material for preclinical studies by transient gene expression (TGE) is addressing this factor in an impressive way: following DNA transfection, the production phase for TGE is usually 2-10 days. Recombinant proteins (r-proteins) produced by TGE can therefore enter the drug development and screening process in a very short time--weeks. With "classical" approaches to protein expression from mammalian cells, it takes months to establish a productive host cell line. This article summarizes efforts in industry and academia to use TGE to produce tens to hundreds of milligrams of r-proteins for either fundamental research or preclinical studies

The influence of the accessory genome on bacterial pathogen evolution

Bacterial pathogens exhibit significant variation in their genomic content of virulence factors. This reflects the abundance of strategies pathogens evolved to infect host organisms by suppressing host immunity. Molecular arms-races have been a strong driving force for the evolution of pathogenicity, with pathogens often encoding overlapping or redundant functions, such as type III protein secretion effectors and hosts encoding ever more sophisticated immune systems. The pathogens’ frequent exposure to other microbes, either in their host or in the environment, provides opportunities for the acquisition or interchange of mobile genetic elements. These DNA elements accessorise the core genome and can play major roles in shaping genome structure and altering the complement of virulence factors. Here, we review the different mobile genetic elements focusing on the more recent discoveries and highlighting their role in shaping bacterial pathogen evolution

Basic Amino Acid Mutations in the Nuclear Localization Signal of Hibiscus Chlorotic Ringspot Virus p23 Inhibit Virus Long Distance Movement

10.1371/journal.pone.0074000PLoS ONE89-POLN

Evaluating the importance of metamorphism in the foundering of continental crust

The metamorphic conditions and mechanisms required to induce foundering in deep arc crust are assessed using an example of representative lower crust in SW New Zealand. Composite plutons of Cretaceous monzodiorite and gabbro were emplaced at ~1.2 and 1.8 GPa are parts of the Western Fiordland Orthogneiss (WFO); examples of the plutons are tectonically juxtaposed along a structure that excised ~25 km of crust. The 1.8 GPa Breaksea Orthogneiss includes suitably dense minor components (e.g. eclogite) capable of foundering at peak conditions. As the eclogite facies boundary has a positive dP/dT, cooling from supra-solidus conditions (T > 950 ºC) at high-P should be accompanied by omphacite and garnet growth. However, a high monzodioritic proportion and inefficient metamorphism in the Breaksea Orthogneiss resulted in its positive buoyancy and preservation. Metamorphic inefficiency and compositional relationships in the 1.2 GPa Malaspina Pluton meant it was never likely to have developed densities sufficiently high to founder. These relationships suggest that the deep arc crust must have primarily involved significant igneous accumulation of garnet–clinopyroxene (in proportions >75%). Crustal dismemberment with or without the development of extensional shear zones is proposed to have induced foundering of excised cumulate material at P > 1.2 GPa

Sarco/Endoplasmic Reticulum Ca2+-ATPases (SERCA) Contribute to GPCR-Mediated Taste Perception

The sense of taste is important for providing animals with valuable information about the qualities of food, such as nutritional or harmful nature. Mammals, including humans, can recognize at least five primary taste qualities: sweet, umami (savory), bitter, sour, and salty. Recent studies have identified molecules and mechanisms underlying the initial steps of tastant-triggered molecular events in taste bud cells, particularly the requirement of increased cytosolic free Ca2+ concentration ([Ca2+]c) for normal taste signal transduction and transmission. Little, however, is known about the mechanisms controlling the removal of elevated [Ca2+]c from the cytosol of taste receptor cells (TRCs) and how the disruption of these mechanisms affects taste perception. To investigate the molecular mechanism of Ca2+ clearance in TRCs, we sought the molecules involved in [Ca2+]c regulation using a single-taste-cell transcriptome approach. We found that Serca3, a member of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) family that sequesters cytosolic Ca2+ into endoplasmic reticulum, is exclusively expressed in sweet/umami/bitter TRCs, which rely on intracellular Ca2+ release for signaling. Serca3-knockout (KO) mice displayed significantly increased aversive behavioral responses and greater gustatory nerve responses to bitter taste substances but not to sweet or umami taste substances. Further studies showed that Serca2 was mainly expressed in the T1R3-expressing sweet and umami TRCs, suggesting that the loss of function of Serca3 was possibly compensated by Serca2 in these TRCs in the mutant mice. Our data demonstrate that the SERCA family members play an important role in the Ca2+ clearance in TRCs and that mutation of these proteins may alter bitter and perhaps sweet and umami taste perception

Earthquake nucleation in the lower crust by local stress amplification

Deep intracontinental earthquakes are poorly understood, despite their potential to cause significant destruction. Although lower crustal strength is currently a topic of debate, dry lower continental crust may be strong under high-grade conditions. Such strength could enable earthquake slip at high differential stress within a predominantly viscous regime, but requires further documentation in nature. Here, we analyse geological observations of seismic structures in exhumed lower crustal rocks. A granulite facies shear zone network dissects an anorthosite intrusion in Lofoten, northern Norway, and separates relatively undeformed, microcracked blocks of anorthosite. In these blocks, pristine pseudotachylytes decorate fault sets that link adjacent or intersecting shear zones. These fossil seismogenic faults are rarely >15 m in length, yet record single-event displacements of tens of centimetres, a slip/length ratio that implies >1 GPa stress drops. These pseudotachylytes represent direct identification of earthquake nucleation as a transient consequence of ongoing, localised aseismic creep

Disulfiram modulated ROS–MAPK and NFκB pathways and targeted breast cancer cells with cancer stem cell-like properties

BACKGROUND: Previous studies indicate that disulfiram (DS), an anti-alcoholism drug, is cytotoxic to cancer cell lines and reverses anticancer drug resistance. Cancer stem cells (CSCs) are the major cause of chemoresistance leading to the failure of cancer chemotherapy. This study intended to examine the effect of DS on breast cancer stem cells (BCSCs). METHODS: The effect of DS on BC cell lines and BCSCs was determined by MTT, western blot, CSCs culture and CSCs marker analysis. RESULTS: Disulfiram was highly toxic to BC cell lines in vitro in a copper (Cu)-dependent manner. In Cu-containing medium (1 mu M), the IC50 concentrations of DS in BC cell lines were 200-500 nM. Disulfiram/copper significantly enhanced (3.7-15.5-fold) cytotoxicity of paclitaxel (PAC). Combination index isobologram analysis demonstrated a synergistic effect between DS/Cu and PAC. The increased Bax and Bcl2 protein expression ratio indicated that intrinsic apoptotic pathway may be involved in DS/Cu-induced apoptosis. Clonogenic assay showed DS/Cu-inhibited clonogenicity of BC cells. Mammosphere formation and the ALDH1(+VE) and CD24(Low)/CD44(High) CSCs population in mammospheres were significantly inhibited by exposure to DS/Cu for 24 h. Disulfiram/copper induced reactive oxygen species (ROS) generation and activated its downstream apoptosis-related cJun N-terminal kinase and p38 MAPK pathways. Meanwhile, the constitutive NF kappa B activity in BC cell lines was inhibited by DS/Cu. CONCLUSION: Disulfiram/copper inhibited BCSCs and enhanced cytotoxicity of PAC in BC cell lines. This may be caused by simultaneous induction of ROS and inhibition of NF kappa B. British Journal of Cancer (2011) 104, 1564-1574. doi: 10.1038/bjc.2011.126 www.bjcancer.com Published online 12 April 2011 (C) 2011 Cancer Research U