FISH was an alternative technique for determining gene amplification and had some distinct advantages over Southern hybridization. Our results demonstrate that HER-2/neu gene amplification in the absence of adjuvant therapy is an independent predictor of poor clinical outcome and is a stronger discriminant than tumor size. Women with small tumors that had gene amplification were at increased risk of recurrence and disease-related death.

Bispecific IgG production in single host cells has been a much sought-after goal to support the clinical development of these complex molecules. Current routes to single cell production of bispecific IgG include engineering heavy chains for heterodimerization and redesign of Fab arms for selective pairing of cognate heavy and light chains. Here, we describe novel designs to facilitate selective Fab arm assembly in conjunction with previously described knobs-into-holes mutations for preferential heavy chain heterodimerization. The top Fab designs for selective pairing, namely variants v10 and v11, support near quantitative assembly of bispecific IgG in single cells for multiple different antibody pairs as judged by high-resolution mass spectrometry. Single-cell and in vitro-assembled bispecific IgG have comparable physical, in vitro biological and in vivo pharmacokinetics properties. Efficient single-cell production of bispecific IgG was demonstrated for human IgG1, IgG2 and IgG4 thereby allowing the heavy chain isotype to be tailored for specific therapeutic applications. Additionally, a reverse chimeric bispecific IgG2a with humanized variable domains and mouse constant domains was generated for preclinical proof-of-concept studies in mice. Efficient production of a bispecific IgG in stably transfected mammalian (CHO) cells was shown. Individual clones with stable titer and bispecific IgG composition for >120 days were readily identified. Such long-term cell line stability is needed for commercial manufacture of bispecific IgG. The single-cell bispecific IgG designs developed here may be broadly applicable to biotechnology research, including screening bispecific IgG panels, and to support clinical development.

Emerging epidemics of hand-foot-and-mouth disease (HFMD) associated with enterovirus 71 (EV71) has become a serious concern in mainland China. It caused 126 and 353 fatalities in 2008 and 2009, respectively. The epidemiologic and pathogenic data of the outbreak collected from national laboratory network and notifiable disease surveillance system. To understand the virological evolution of this emerging outbreak, 326 VP1 gene sequences of EV71 detected in China from 1987 to 2009 were collected for genetic analyses. Evidence from both traditional and molecular epidemiology confirmed that the recent HFMD outbreak was an emerging one caused by EV71 of subgenotype C4. This emerging HFMD outbreak is associated with EV71 of subgenotype C4, circulating persistently in mainland China since 1998, but not attributed to the importation of new genotype. Originating from 1992, subgenotype C4 has been the predominant genotype since 1998 in mainland China, with an evolutionary rate of 4.6∼4.8×10−3 nucleotide substitutions/site/year. The phylogenetic analysis revealed that the majority of the virus during this epidemic was the most recent descendant of subgenotype C4 (clade C4a). It suggests that the evolution might be one of the potential reasons for this native virus to cause the emerging outbreak in China. However, strong negative selective pressure on VP1 protein of EV71 suggested that immune escape might not be the evolving strategy of EV71, predicting a light future for vaccine development. Nonetheless, long-term antigenic and genetic surveillance is still necessary for further understanding.

Phycobiliproteins, unlike other light-harvesthmg proteins involved in photosynthesis, bear covalently attached chromophores. The bilin chromophores are attached through thioether bonds to cysteine residues. The cyanobacterium Synechococcus sp. PCC 7002 has eight distinct blin attachmnt sites on seven poypeptides, all ofwhich carry the same chromophore, phycocyanobilin. When two genes in the phycocyanin operon of this organisn, cpcE and cpcF, are inactivated by insertion, together or separately, the srping result is elimination of correct biln attachment at only one site, that on the a subunit of phycocyanin. We have overproduced CpcE and CpcF in Escherichia coli. In vitro, these proteins catalyze the attachment of phycocyanobilin to the a subunit of apophycocyanin at the appropriate site, a-Cys-84, to form the correct adduct. CpcE and CpcF also efficiently catalyze the reverse reaction, in which the bilin from holo-a subunit is transferred either to the apo-a subunit of the same C-phycocyanin or to the apo-a subunit of a heterologous C-phycocyanin. The forward and reverse reactions each require both CpcE and CpcF and are specific for the a-Cys-84 position. Phycocyanobilin is the immediate precursor of the protein-bound bilin.Phycobiliproteins are homologous proteins found in cyanobacteria, red algae, and the cryptomonads (1). In vivo they form highly ordered macromolecular light-harvesting assemblies, phycobilisomes, in which directional energy transfer is determined by the spectroscopic properties and relative positions of the bilin prosthetic groups.The number of bilin attachment sites on all of the phycobiliproteins ofa particular cyanobacterium or red alga ranges from a minimum of 8, all carrying the same bilin, to >20, with up to three different bilins. To study the process of bilin attachment we have chosen a simple case, that in Synechococcus sp. PCC 7002, in which there are 8 attachment sites for the same bilin, phycocyanobilin (PCB), on seven polypeptides.Posttranslational modification of proteins usually involves recognition of simple amino acid sequence determinants. Local sequence homology around modification sites generally suggests that there is one enzyme that recognizes all of these sites. Surprisingly, this does not appear to be the case for bilin attachment to phycobiliproteins.Until recently little was known about the processes of bilin synthesis and attachment to the apoproteins. Beale and Comejo (2-4) have examined bilin biosynthesis in the red alga Cyanidium caldarium and have provided evidence for the following pathway: heme --biliverdin IXa -15,16-dihydrobiliverdin IXa -. 3(Z)-phycoerythrobilin -3(Z)-PCB -+ 3(E)-PCB.PCB can be obtained by cleavage from phycocyanin or allophycocyanin by methanolysis (5). PCB has an ethylidene group at the C-3 position (the IUPAC numbering system for bilins is shown for PCB in ref. 2) and is the product of elimination of cysteine at the 3' carbon of the protein-bound bilin (3,3'-dihydro-3'-cysteinylphycocyanobilin; referred to below as PCB adduct).T...