Unfinished business: an essay on finally leaving the bench

Spontaneous and induced chromosome aberrations have been studied over more than a century. The resolution of detection of aberrations has depended on the improvement of available techniques. An overview on the major high lights in this area of research, from the time of solid staining to fluorescence in situ hybridization technique is presented in this review.     

Unfinished business: Incomplete laminin processing exposes a tumor target

The G45 domain of laminin-332 is cleaved in normal tissues but remains in squamous cell carcinomas. Targeting this domain using a G45 antibody reduced in vivo tumor growth and invasion and suggests a role for G45 as a new therapeutic target.     

Unfinished business: Incomplete laminin processing exposes a tumor target

The G45 domain of laminin-332 is cleaved in normal tissues but remains in squamous cell carcinomas. Targeting this domain using a G45 antibody reduced in vivo tumor growth and invasion and suggests a role for G45 as a new therapeutic target.Key words: laminin, extracellular matrix, cancer, squamous cell carcinoma, invasionLaminin-332 (previously known as laminin-5) is a trimeric extracellular glycoprotein important for the integrity of the basement membrane zone.¹ It is found at high levels in squamous cell carcinomas (SCC) and its expression correlates with increased tumor invasiveness and poor prognosis.² In a recent article in Cancer Research, Marinkovich and colleagues³ show that the G45 domain of laminin-332, normally removed by proteolytic processing, remains present in SCC tissue.To determine the function of the G45 domain, the authors expressed the wild-type α3 chain of laminin-332 or the α3 chain lacking G45 (ΔG45) in normal and transformed laminin-332-null keratinocytes. They then examined whether the phenotype of the G45 deletion mutant could be reverted by re-expressing the G45 domain peptide.When expressed in laminin-332-null keratinocytes, the wild-type form of the protein was found deposited in the extracellular matrix. Reduced matrix deposition was observed with the ΔG45 mutant. In addition, the ΔG45 mutant was associated with abnormal peripheral cell adhesions, in contrast to central α6 integrin-associated adhesions in the wild-type. ΔG45 cells detached more rapidly in trypsin and showed increased cell migration in vitro. Expression of the G45 domain peptide restored laminin-332 deposition and reversed the changes in cell adhesion and migration.After transformation with Ras and IκBα, laminin-null keratinocytes are tumorigenic and invade Matrigel. Expression of wild-type laminin-332 increases invasiveness in these cells. Invasion was also increased, but to a lesser degree, with the ΔG45 mutant expressing cells. ΔG45 cells had reduced MMP levels, and both invasion and MMP production were restored by expression of the G45 domain. Similar results were seen in an in vivo SCC model where reduced tumor growth and muscle invasion correlated with expression of the ΔG45 mutant relative to wild-type protein. Interestingly, activation of the PI3K pathway almost completely restored tumor growth and laminin-332 deposition in the ΔG45 mutant expressing cells. In vitro, ΔG45 cells show decreased Akt phosphorylation and phospho- ERK nuclear translocation. Both could be corrected by activating PI3K. Together, these results suggest that G45 may promote laminin- 332 deposition via a signaling mechanism rather than acting simply as a matrix anchor.As G45 promotes tumorigenesis in SCC and is absent in normal tissues, could it be a target for anti cancer therapy in vivo? The authors show that an antibody targeting G45 dramatically inhibited subcutaneous SCC tumor growth. Intriguingly, despite recognizing both G45 and native laminin-332 by western blot, the antibody caused no disruption of epithelial-mesenchymal integrity in normal tissues.This study has shown an important role for the G45 domain of laminin-332 in SCC tumorigenesis. Considering its absence in normal tissues, G45 represents a potential target for anti-cancer therapy in human SCC.     

Unfinished business: efforts to define dual-use research of bioterrorism concern

Biotechnological research poses a special security problem because of the duality between beneficial use and misuse. In order to find a balance between regulating potentially dangerous research and assuring scientific advancement, a number of assessments have tried to define which types of research are especially open to misuse and should therefore be considered dual-use research of special concern requiring rigorous oversight. So far, there has been no common understanding of what such activities are. Here we present a review of 27 assessments focusing on biological dual-use issues published between 1997 and 2008. Dual-use research activities identified by these assessments as being of special concern were compiled and compared. Moreover, from these 27 assessments, the primary research publications explicitly identified as examples of concerning research activities were extracted and analyzed. We extracted a core list of 11 activities of special concern and show that this list does not match with the reasons why primary research publications were identified as being of special concern. Additionally, we note that the 11 activities identified are not easily conducted or replicated, and therefore the likelihood of their being used in a high-tech mass casualty bioterrorism event should be reevaluated.     

Fleming's Unfinished

It is often suggested that, soon after he discovered penicillin, Alexander Fleming lost interest in what became the most important of all the antibiotics. Fleming's notebooks, however, show that he continued working with penicillin throughout the 1930s, even to the point when Florey and Chain became interested in it. During this period, Fleming isolated new airborne molds and checked them for their ability to produce antibacterial agents, and he also investigated other examples of microbial antagonism, such as bacteriophages. Unfortunately, none of this work was published. What follows is a simulation, based largely upon Fleming's notebooks, of a paper that he might have written in early 1940. This version of "Fleming's Unfinished" should once and for all dismiss the view that he failed fully to recognize the significance of his famous discovery.     

The Unfinished Story of Cytochrome f

Cytochrome f is a unique, integral membrane protein. The background to its discovery by Robert Hill (1899-1991) and Ronald Scarisbrick over 60 years ago and the influence of David Keilin (1887-1963) and Frederick Gowland Hopkins (1861-1947) are discussed. The development of methods for isolating cytochrome f is outlined, emphasizing the remarkable achievement of Hill and Scarisbrick at a time when few if any membrane proteins had been isolated, and the importance of the discovery of a natural proteolysis in Brassica spp., stimulated by organic solvents, by Eijiro Yakushiji and coworkers and by Masa-aki Takahashi and Kozi Asada in 1975. The significance of different types of instrumentation in the study of cytochrome f is discussed, drawing attention to the importance of the microspectroscope ocular for its discovery, to types of spectrophotometer developed especially by Britton Chance for spectrophotometric measurements on turbid suspensions of cells and plastids, and to the history of stopped-flow spectrophotometry. The stopped-flow instrument originated in the bucket-scale flow methods of Hartridge and Roughton (1923), and was later developed on the microscale by Chance. Finally, the problems that remain for understanding the behavior of cytochrome f in the thylakoid lumen are contrasted with the significance of in vitro studies that provide a paradigm for transient protein-protein interactions in the wider field of biology as a whole.     

Unfinished Stories on Viral Quasispecies and Darwinian Views of Evolution

Experimental evidence that RNA virus populations consist of distributions of mutant genomes, termed quasispecies, was first published 31 years ago. This work provided the earliest experimental support for a theory to explain a system that replicated with limited fidelity and to understand the self-organization and adaptability of early life forms on Earth. High mutation rates and quasispecies dynamics of RNA viruses are intimately related to both viral disease and antiviral treatment strategies. Moreover, the quasispecies concept is being applied to other biological systems such as cancer research in which cellular mutant spectra can be also detected. This review addresses some of the unanswered questions regarding viral and theoretical quasispecies concepts as well as more practical aspects concerning resistance to antiviral treatments and pathogenesis.