A new knockout human cell line has helped researchers make some huge leaps in the discovery of causative factors of some infectious diseases. The human cell line has been developed with only one copy of thousands of genes (chromosome 8 remained diploid), allowing for knockouts to be generated in which there isn't a second copy of the gene to compensate for the loss, through transcription of the encoded protein. The cell line, generated by a group from the Whitehead Institute for Biomedical Research, was used to study which genes are used by both viral and bacterial pathogens, one of which was the influenza virus. In a November 27 article in Science, the team describes which host factors (genes and gene products) are required for influenza infection and cytotoxic effects of several bacterial toxins.

Biosimilars are follow-on biopharmaceuticals, made by competitors of a branded drug, following expiry of the patent. By definition, they tend to be large molecules, as opposed to small molecule drugs or therapies, like siRNA. Being more complex, their production can also be a complicated process, and since the manufacturer might not have information such as exact culture fermentation conditions, or access to transgenic organisms used to produce the original product, biosimilar manufacturing processes are not identical. This gives rise to the potential for unprecedented and unpredictable problems such as immunogenic (allergic) reactions to impurities and breakdown products. Thus, unlike generic chemical drugs, the use of biosimilars can pose serious health risks.

The European Union has a system in place for approving biosimilars, but the USA has not yet followed suit. However, it is widely accepted that generic drug approval pathways are not appropriate for complex biologics, and a proposal for a pathway for biosimilars approval was made earlier this year.

From a business perspective, biosimilars may or may not be lucrative investments, depending on how safe they are, how easily they are approved and whether practitioners and the public are willing to accept them. For those working in this area of biopharmaceuticals, or interested in biosimilars, a comprehensive review of biosimilars regulation and market can be purchased online from Espicom Business Intelligence.

The internet is full of fun tools for enhancing your knowledge of biochemistry and biotechnology. One such resource is the Amino Acid Explorer, on the NCBI website. Students in introductory biotechnology courses might find the Structure and Chemistry button helpful for learning functional groups of amino acids and their properties. If you're in a more advanced program, check out the Amino Acids At Work button for the roles of different amino acids in the active sites of proteins, and the Mutation Analyzer button lets you look up the effects of mutations, or could be useful for investigating possible gene polymorphisms. The Amino Acids As Ligands button takes you to a library of 3-D pictures of protein-ligand complexes with links to the articles describing their role. For example, I checked out the role of a ligand containing valine in the biosynthesis of penicillin.

A number of global initiatives in recent years, including ICH and PAT, have encouraged the use of single-use systems in bioprocessing. A major benefit to adapting process systems in this way is the reduced risk of cross-contamination between batches. Traditionally, single-use systems may have been used in early stage development of a new product, but commercial production would be converted to the use of stainless steel, multiple-use systems. Improvements to culturing techniques and optimization of fermentation conditions have made it possible for companies to achieve better titers, and, thus, able to use smaller bioreactors, which has contributed to increased use of disposible equipment. Controversy is beginning to brew, however, over the use of single-use systems, since focus has begun to shift from how to design systems with disposibles to what the environmental impacts may be. If you're curious about who is using single-use systems, and for what purposes, BioProcess International has published the results of a survey on the Impact of Single-Use Technologies, online.

_{Source: McLeod, L. 2009. Advances in Bioprocessing. BioProcess International, May 2009.}

The fruit fly, (Drosophila), has been used as a genetic research tool for a very long time, because of it's easily observable phenotypic differences, and adherence of many of these traits to the basic rules of Mendelian inheritance. Because of this, I always assumed, or thought I'd heard somewhere, that it was the first multicelled organism for which the entire DNA sequence was determined. This is wrong, however, and, according to the NCBI Nematode Genome Resources webpage, the nematode was the first multicellular eukaryote to have it's genome completely sequenced.

This information was bought to my attention during a presentation at the SETAC conference I am attending, on the use of genomics and DNA microarray techniques to study nanoparticle toxicity using silver nanoparticles (Ag NP). The soil nematode is a popular test organism for toxicity studies. The Ag NP were found to severely downregulate several genes in this organism, by affecting transcription. This paper provided further support for the use of the nematode as a bioindicator organism for environmental contamination, using proteomic and genomic techniques.

_{Source: Roh, JY. et al. 2009. Ecotoxicity of silver nanoparticles on the soil nematode Caenorhabditis elegans using functional ecotoxicogenomics. Environmental Science and Technology 43:3933-3940.}

While the industrial biotechnology sector is rushing full steam ahead to develop and commercialize new nanotechnology products, scientists working in areas of human and ecological toxicity are scrambling to keep up. Nanoparticle toxicity is a prominent topic at this year's SETAC NA conference, with several dedicated platform and poster sessions. The society is primarily concerned with environmental toxicology, rather than human, but the number of papers, and the variety of different types of nanoparticles and target organisms being investigated, is reassuring. I'm glad to see the potential dangers of nanoparticle toxicity are being taken seriously by industry and regulators alike. Here are some of the presentations I came across:

• Kovacs, T. et al. Ensuring the Environmental Compatibility of Manufactured Nanocrystalline Cellulose (NCC). NCC is being developed for the pulp and paper industry. Four aquatic species were tested to determine possible adverse effects of an accidental release. Both acute and subacute endpoints were measured and conclusions were that NCC is of low risk to the aquatic environment.
• Ahamed, M. et al. Silver Nanoparticles-Induced Heat Shock Protein 70, Oxidative Stress and Apoptosis in Drosophila melanogaster. Silver nanoparticles (Ag NP) are already in commercial use in many different forms. There were a number of presentations on their uptake, accumulation and toxicity. This paper reported the induction of a number of enzymes involved in apoptosis, DNA repair, reactive oxygen species scavenging and a number of other response mechanisms to cellular stressors. In another presentation (Shoults-Wilson, W. et al. The Influence of Particle Size, Concentration and Surface Functionalization on Bioavailability of Ag Nanoparticles in the Earthworm Eisenia fetida), earthworms were found to avoid soil containing Ag NP. Bioaccumulation of Ag NP was observed, and exposure resulted in a dose-dependant reduction in reproduction.
• Roh, J.Y. et al. Toxicological Investigation of Multi-Well Carbon Nanotubes in Caenorhabditis elegans Using Genomics and Proteomics Approach. The soil nematode was exposed to multi-well carbon nanotubes (MW-CNT) and toxicity investigated using endpoints such as mortality, growth and reproduction, in addition to whole genome microarray and proteomics methods. Although mortality was not significantly affected, reproductive potential was reduced after exposure to the MW-CNT. A number of genes were differentially expressed including those for heat-shock proteins, suggesting the organisms were under stress. Work from the same research team also reported for Ag NP, for which toxicity was measured using this organism and an ecotoxicogenomic approach (Park, YJ and Choi, J. A Toxicity Assay Using Stress Responsive Caenorhabditis elegans Mutant Strains).
• Kim, J. et al. Ultraviolet B Light Enhances Toxicity of CdSe/ZnSe Quantum Dots in Daphnia magna. The results of this study suggest that phototoxicity is mediated by both release of Cd from the quantum dot (QD), and production of reactive oxygen species on the QD surface.

Developments in proteomics and genomics make it possible to study the impacts of environmental contaminants on the expression of hundreds, even thousands, of genes. 2-Dimensional Gel Electrophoresis is a protein separation method used in proteomics, for partitioning proteins from a mixture according to two criteria, instead of just one. Traditional gel electrophoresis, usually applied to the separation of DNA, works in just one direction.

In a presentation today, at the SETAC North America conference (New Orleans, LA), Jennifer Cole, of Texas Tech University, described using proteomics to study the effects of benzo(a)pyrene (B(a)P), a potent carcinogen, on protein expression in the blubber of the Bottlenose Dolphin. The work is the first of its kind on a marine mammal. Skin and blubber cultures from freshly stranded dolphins, were treated with B(a)P which is a known contaminant in marine environments. Proteins were extracted, desalted and separated on a 2-D gel. The gels were run in one direction to separate the proteins based on isoelectric point (the pH at which the sum of charges of the amino acids is zero), and in a second direction to separate based on molecular weight. Using the gels, the team could also apply protein purification methods to isolate and identify specific enzymes and determine which ones were up regulated and which were down regulated, as a result of exposure to the PAH compound

Biomarkers are an important tool used by toxicologists and environmental scientists, to study environmental exposures of plants and animals to potentially toxic compounds. Many of the methods for studying gene expression and enzyme activity, attributed to biotech and biomedical research, are also applicable to environmental studies. I am privileged to be able to attend the SETAC North America conference in New Orleans (LA) this week. While on the lookout for new information on ecological risk assessments, I've also come across a number of examples of how genetic methods are being applied to bioindicator discovery.

In one example, quantitative reverse-transcriptase (RT) PCR was used to study Vitellogenin (Vg) gene expression in male fathead minnows exposed to estrogenic substances. Vg is an egg-yolk precursor protein usually only expressed in female fish. The gene is dormant in males, but is expressed when they have been exposed to estrogenic compounds (chemicals that mimic the effects of the hormone estrogen). Reddy et al., of the US EPA, purified total RNA from the minnows and subjected the samples to reverse transcription using a retrovirus reverse transcriptase enzyme, producing cDNA, which was then quantified by PCR. Vg gene expression was altered at concentrations below routine laboratory detection limits, for three of the four compounds tested. Therefore, measurements of Vg might provide a good biomarker of exposure to estrogenic compounds, where chemical analysis is not sensitive enough.

_{Source: Reddy, TV et al. Induction of vitellogenin gene expression in adult male fathead minnows for select estrogens in 48-hr continuous addition and daily renewal exposures. Presented at SETAC NA, New Orleans, Nov. 2009.}

A tissue is an aggregate of cells, growing and thriving in an environment where they adhere and interact with one another. Tissue Engineering is the use of bioengineering methods to create, improve, develop and grow tissues, which then may be used for grafting, cartilage repair or, ultimately, regenerative medical procedures. The study of tissues is aimed at determining the answers to fundamental questions such as how cells react and interact in a specific matrix, and may involve the use of proteomics to study gene expression and protein production in complex environments. This form of systems biology might look at cellular functions such as excretion of intercellular signaling substances, and epigenetic factors that determine physical features such as size and shape of organs.

One of the goals of tissue engineers is to reverse the effects of injury or aging of cartilage, nerve damage or scarring from burns and other trauma. Another major achievement would be the ability to grow entire organs using stem cells taken from the organ recipient, replacing the need for a suitable donor and eliminating the risk and waiting time required for finding said donor, or the potential complications of xenotransplantation.

One breakthrough in tissue engineering announced in 2009 was the development of a biodegradable resin, by a group at University of Twente, that could be used as a scaffold in situ, for human organ generation. Another significant development, announced by scientists at Stanford and NY University School of Medicine, was the ability to grow tissues in an environment of well-vascularized cells. Using laboratory animals, they demonstrated that fatty tissue from the groin area of rats containing blood vessels, fat and skin, could be used as a scaffold for stem cells. They called the scaffolds "explanted microcirculatory beds" (EMBs). Once the cells are well embedded in the tissue they can be transplanted back into the animal, where they are not rejected. Both of these discoveries hold enormous promise for therapeutic cloning and organ transplant research.

_Sources:

InSciences Organisation. Breakthrough in Twente: Biodegradable synthetic resin replaces vital body parts. 13 June 2009.

Chang et al. 2009. Tissue engineering using autologous microcirculatory beds as vascularized bioscaffolds. FASEB Journal, March 2009. doi:10.1096/fj.08-114868.

There are many challenges to making stem cell therapies such as regenerative medicine actually work in a therapeutic setting. We might be able to harvest stem cells, from either blastocysts or by creating pluripotent cells from already differentiated tissues, but that's really only the beginning of a medically viable process. Once a cell line is cultured in a maintainable way, the following questions remain:

• How to direct differentiation into the desired tissue type
• Optimizing growth conditions and the physical environment for cell cultures or for growing organs for transplant
• How to inject and transport stem cells to the target location in the body
• Finding ways to generate IPSCs without inducing tumor formation in future recipients of stem cell therapies