Safety,security, and serving the public interest in synthetic biology

This article describes what may be done by scientists and by the biotechnology industry, generally, to address the safety and security challenges in synthetic biology. Given the technical expertise requirements for developing sound policy options, as well as the importance of these issues to the future of the industry, scientists who work in synthetic biology should be informed about these challenges and get involved in shaping policies relevant to the field.     

Change of cytokeratin filament organization during the cell cycle: selective masking of an immunologic determinant in interphase PtK2 cells

The organization of intermediate-sized filaments (IF) of the cytokeratin type was studied in cultures of PtK2 cells in which typical IF structures are maintained during mitosis, using a monoclonal antibody (KG 8.13). This antibody reacts, in immunoblotting experiments, with the larger of the two major cytokeratin polypeptides present in these cells but, using standard immunofluorescence microscopy procedures, does not react with the cytokeratin filaments abundant in interphase cells, in striking contrast to various antisera and other monoclonal cytokeratin antibodies. In the same cell cultures, however, the antibody does react with cytokeratin filaments of mitotic and early postmitotic cells. The specific reaction with cytokeratin filaments of mitotic cells only is due to the exposure of the specific immunologic determinant in mitosis and its masking in interphase cells. Treatment of interphase cells with both Triton X-100 as well as with methanol and acetone alters the cytokeratin filaments and allows them to react with this monoclonal antibody. A similar unmasking was noted after treatment with buffer containing 2 M urea or low concentrations of trypsin. We conclude that the organization of cytokeratin, albeit still arranged in typical IF, is altered during mitosis of PtK2 cells.     

Properties of gallic acid-induced extracellular laccase of Botrytis cinerea

The properties of the extracellular laccase produced by Botrytis cinerea induced by garlic acid are compared with the laccase produced by Botrytis in the presence of grape juice. The extra- and intracellular laccases are compared and found to be very similar.     

Graft versus neuroblastoma reaction is efficiently elicited by allogeneic bone marrow transplantation through cytolytic activity in the absence of GVHD

Continuous efforts are dedicated to develop immunotherapeutic approaches to neuroblastoma (NB), a tumor that relapses at high rates following high-dose conventional cytotoxic therapy and autologous bone marrow cell (BMC) reconstitution. This study presents a series of transplant experiments aiming to evaluate the efficacy of allogeneic BMC transplantation. Neuro-2a cells were found to express low levels of class I major histocompatibility complex (MHC) antigens. While radiation and syngeneic bone marrow transplantation (BMT) reduced tumor growth (P < 0.001), allogeneic BMT further impaired subcutaneous development of Neuro-2a cells (P < 0.001). Allogeneic donor-derived T cells displayed direct cytotoxic activity against Neuro-2a in vitro, a mechanism of immune-mediated suppression of tumor growth. The proliferation of lymphocytes from congenic mice bearing subcutaneous tumors was inhibited by tumor lysate, suggesting that a soluble factor suppresses cytotoxic activity of syngeneic lymphocytes. However, the growth of Neuro-2a cells was impaired when implanted into chimeric mice at various times after syngeneic and allogeneic BMT. F1 (donor-host) splenocytes were infused attempting to foster immune reconstitution, however they engrafted transiently and had no effect on tumor growth. Taken together, these data indicate: (1) Neuro-2a cells express MHC antigens and immunogenic tumor associated antigens. (2) Allogeneic BMT is a significantly better platform to develop graft versus tumor (GVT) immunotherapy to NB as compared to syngeneic (autologous) immuno-hematopoietic reconstitution. (3) An effective GVT reaction in tumor bearing mice is primed by MHC disparity and targets tumor associated antigens.     

Application of purging biotinylated liposomes from plasma to elucidate influx and efflux processes associated with accumulation of liposomes in solid tumors

Although small, 100-nm liposomes are known to selectively accumulate in solid tumors, the individual contributions of liposome influx and egress rates are not well understood. The aim of this work was to determine influx and efflux kinetics for 100-nm, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/cholesterol (Chol) liposomes by inducing aggregate formation of biotinylated liposomes upon administering avidin. Injecting 50 microg of neutravidin intravenously to mice that had previously been administered 100 mg/kg DPSC/Chol liposomes containing 0.5 mol% biotin-conjugated lipid resulted in >90% elimination of the liposomes from plasma within 1 h. This rapid removal by the reticuloendothelial system (RES) permitted the determination of the tumor efflux kinetics due to negligible tumor influx after neutravidin injection. The tumor efflux rate constant (k(-1)) was determined to be 0.041 h(-1) when neutravidin was injected 4 h after liposome injection. This allowed the determination of the tumor influx rate constant (k(1)), which under these conditions was 0.022 h(-1). Therefore, DSPC/Chol liposomal accumulation, in LS180 solid tumors, is dictated primarily by plasma liposome concentrations and liposome egress is comparable or slightly faster than influx into the tumors. This method is applicable for a wide range of lipid doses, and can be used to characterize influx and efflux parameters at different time points after accumulation. The application, therefore, has the potential to be used to fully characterize the impact of different liposome parameters such as lipid composition, steric stabilization, size and dose on tumor accumulation kinetics.     

Detection of a cytokeratin determinant common to diverse epithelial cells by a broadly cross-reacting monoclonal antibody.

A monoclonal antibody derived from a mouse immunized with bovine epidermal prekeratin has been characterized by its binding to cytoskeletal polypeptides separated by one- or two-dimensional gel electrophoresis and by immunofluorescence microscopy. This antibody (KG 8.13) binds to a determinant present in a large number of human cytokeratin polypeptides, notably some polypeptides (Nos. 1, 5, 6, 7, and 8) of the 'basic cytokeratin subfamily' defined by peptide mapping, as well as a few acidic cytokeratins such as the epidermis-specific cytokeratins Nos. 10 and 11 and the more widespread cytokeratin No. 18. This antibody reacts specifically with a wide variety of epithelial tissues and cultured epithelial cells, in agreement with previous findings that at least one polypeptide of the basic cytokeratin subfamily is present in all normal and neoplastic epithelial cells so far examined. The antibody also reacts with corresponding cytokeratin polypeptides in a broad range of species including man, cow, chick, and amphibia but shows only limited reactivity with only a few rodent cytokeratins. The value of this broad-range monoclonal antibody, which apparently recognizes a stable cytokeratin determinant ubiquitous in human epithelia, for the immunohistochemical identification of epithelia and carcinomas is discussed.     

High-fidelity KKH variant of Staphylococcus aureus Cas9 nucleases with improved base mismatch discrimination

The Cas9 nuclease from Staphylococcus aureus (SaCas9) holds great potential for use in gene therapy, and variants with increased fidelity have been engineered. However, we find that existing variants have not reached the greatest accuracy to discriminate base mismatches and exhibited much reduced activity when their mutations were grafted onto the KKH mutant of SaCas9 for editing an expanded set of DNA targets. We performed structure-guided combinatorial mutagenesis to re-engineer KKH-SaCas9 with enhanced accuracy. We uncover that introducing a Y239H mutation on KKH-SaCas9’s REC domain substantially reduces off-target edits while retaining high on-target activity when added to a set of mutations on REC and RuvC domains that lessen its interactions with the target DNA strand. The Y239H mutation is modelled to have removed an interaction from the REC domain with the guide RNA backbone in the guide RNA-DNA heteroduplex structure. We further confirmed the greatly improved genome-wide editing accuracy and single-base mismatch discrimination of our engineered variants, named KKH-SaCas9-SAV1 and SAV2, in human cells. In addition to generating broadly useful KKH-SaCas9 variants with unprecedented accuracy, our findings demonstrate the feasibility for multi-domain combinatorial mutagenesis on SaCas9’s DNA- and guide RNA- interacting residues to optimize its editing fidelity.