Category Archives: Biotechnology

Biotechnology

Serial dilution woes

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A recent report adds further evidence that assays relying on serial dilution and tip-based dispensing could be a source of irreproducibility, particularly in pharmacological assays.

A few days after I wrote the methagora entry below about our efforts to improve the reproducibility of published research, somebody pointed out a paper published last week in PLOS ONE that compared the results of automated serial dilution and plastic tip-based dispensing using a robotic sample processor to results obtained by an acoustics-based liquid dispenser. The latter is a technique using sound for noncontact liquid dispensing and is implemented in instruments such as those sold by Labcyte Inc., the employer of one of the authors on the manuscript. The dose-response data comparing the results of these two liquid handling methods, however, was previously published in patents by AstraZeneca on pyrimidine derivatives for inhibiting Eph receptors. The AstraZeneca results showed that data obtained on the 14 reported compounds via acoustic dispensing showed activities that were 1.5 to 276.5 times higher than data coming from serial dilution and tip-based dispensing.

What the PLOS ONE authors added to this story, besides promoting the research results to the press, was the computation of pharmacophores based solely on the two sets of activity data. The pharmacophore computed from the acoustic data was structurally similar to pharmacophores computed from x-ray crystallography data (for example, all these compounds contained hydrophobic binding domains) and was able to predict the activity of subsequent chemicals. In contrast, the pharmacophore computed from the serial dilution and tip-based dispensing data was very different, contained no hydrophobic domains, and was non-predictive.

What should one make of this? Well, it seems logical that hydrophobic domains could influence the results of serial dilution and dispensing through plastic tips via adsorptive or other effects. As one person commenting on the PLOS ONE paper states, such effects have been well documented and proper analytical technique calls for experiments to detect them.

This all reminds me of marketing for HP’s high performance dispenser that also forgoes serial dilution and instead uses inkjet printing technology to dispense undiluted reagents, presumably also via acoustics. HP promotes the increased reliability of this technique for generating dose-response curves but they don’t highlight the kind of effect documented by the authors of the PLOS ONE paper.

If these results are indicative of differences observed between these two types of liquid dispensing it seems that drug companies must be aware of them and are adapting their assays and protocols as necessary. But even if this is the case, there appears to be little evidence that academic researchers are worried about this.

In theory, one can certainly see the appeal of contactless dispensing but more hard data is needed to draw firm conclusions. This will require extensive side-by-side testing of different sample dispensing methods with many different compounds.

At a minimum, researchers need to be cognizant of this potential problem and report how they dispensed their reagents when reporting results from these kinds of pharmacological assays. Better yet, they should repeat key experiments on different days and with different equipment.

Update: I just found out that Derek Lowe has a nice post about this paper over at In the Pipeline

DNA origami on the rise

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Nanotechnology is all the rage these days but its use by practicing biologists is still very limited. A recent entry in the nanotechnology arena is DNA origami, a method for creating nanostructures out of DNA that is more accessible than previous methods and allows larger and more complex structures to be created with greater ease.

In the April issue of Nature Methods you will find a primer to DNA origami that provides an excellent introduction to this technology with valuable practical advice on designing and synthesizing DNA nanostructures using the DNA origami methodology. We hope that this primer will stimulate biologists or others new to this field to take a look at this technology and dream up exciting new applications.

One of the crucial steps of DNA origami is isolating your properly folded structure. A Correspondence by William Shih, one of the pioneers of DNA origami, describes some simple but very useful modifications to an agarose gel electroelution method that many people use for isolating PCR products or small DNA fragments from restriction digests. These changes greatly increase the efficiency of isolating intact large DNA nanostructures compared to existing methods.

Finally, the Editorial discusses the prospects of DNA nanostructures created using DNA origami as biological research tools.

Based on the number of posters describing applications of DNA origami at the 2010 Gordon Research Conference on Single Molecule Approaches to Cell Biology, compared to previous years the biological community and the single molecule biophysics community in particular is showing interest in the methodology. Only time will tell if it fairs better among biologists than other promising nanotechnology tools and methods.

We’d like to know what our readers think of the biological research prospects of this technology, or other nanotechnology tools and methods for that matter. Tell us what you think.

Viola Vogel

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Swiss Federal Institute of Technology, Zurich

A bioengineer discusses how mechanical forces in tissues may promote malignancy.

The connective-tissue protein collagen has been considered to be a structural barrier against tumour invasion in tissues. Enzymes that cleave collagen and other extracellular matrix (ECM) molecules were thus thought to promote tumour progression, but inhibitors of these enzymes have failed in clinical trials. And paradoxically, increased collagen expression is associated with a greater incidence of cancer spread.

Working with mice, Valerie Weaver of the University of California, San Francisco, and her team show that other ECM-remodelling parameters regulate malignancy (K. R. Levental et al. Cell 139, 891–906; 2009). They studied an enzyme that initiates collagen crosslinking and is often found in tissue around tumours. They reveal that the crosslinking increases the stiffness of collagen matrices, which upregulates growth-factor signalling and breast malignancy. This suggests that tumour progression depends on a tissue-remodelling process that is regulated by biochemical and mechanical factors.

Bioengineers developing implantable materials that promote tissue regeneration can also learn a lot from this paper. Dense collagen capsules typically form around implanted biomaterials, which has prompted a search for clues to how to engineer surfaces that promote blood-vessel formation and tissue regeneration rather than scarring.

Knowing which factors promote malignancy may also help us to engineer materials and tissues that tip the balance towards enhanced tissue regeneration. This paper might thus stimulate ideas on how to interfere with the interplay between ECM-crosslinking enzymes that enhance matrix stiffness and ECM-protein-cleaving enzymes. Doing so may affect mechanosensitive cell-signalling pathways, promoting regeneration.

Drew Endy

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Massachusetts Institute of Technology, Cambridge, USA

A biological engineer searches for simplicity.

Several years ago, a good colleague suggested that I read about a discussion held in 1864 on nuts and bolts (J. Franklin Inst. 77, 344–351; 1864). The focus was a paper by one William Sellers that argued for the adoption of a uniform system of screw threads — 60° angles, squared off along the edges.

Machinists across the United States eventually started producing nuts and bolts according to Sellers’ scheme. As a result, hardware stores now offer a wide selection of standardized parts that can be used in combination and behave as expected.

Inspired by this example and others, I have been studying how synthetic biological parts might be made as regular and easy to use as Sellers’ nuts and bolts.

The starting complexity of nature has led some distinguished researchers to doubt such work is practical. But given that there has been little research on manufactured bio-simplicity, this seems premature.

And there are examples: a team at the California Institute of Technology in Pasadena recently developed a uniform system for engineering simple biological switches made from ribonucleic acids (M. N. Win and C. D. Smolke Proc. Natl Acad. Sci. USA doi:10.1073/pnas.0703961104; 2007).

The ‘nuts and bolts’ of the switches are RNA sensor and actuator domains. The method for combining any sensor domain to an actuator domain through a third communication domain provides the ‘uniform screw threads’. Because such switches are produced by a standard process, many switches could be quickly programmed to control diverse cellular functions in response to myriad molecular inputs, from small molecules, to peptides, to nucleic acids.

I suspect that further efforts to engineer biological simplicity will have similarly powerful results.

Robert Langer

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Massachusetts Institute of Technology, USA

A bioengineer sees a future for safe gene-silencing therapies.

The possibility of treating genetic disorders by modifying gene expression has been an attractive yet elusive goal for decades. Problems with the safety and efficacy of various types of gene therapy have held back progress. In particular, there have been some high-profile failures, including a number of deaths during clinical trials.

But seminal studies reported by Andrew Fire and Craig Mello in 1998 led to a potentially new class of therapeutic agent. These researchers, who went on to share a Nobel prize for their work, found that small pieces of RNA, dubbed siRNAs, can silence genes.

Although switching off genes may have fewer complications than adding new ones, the safe and effective delivery of genetic agents remains a critical challenge. I was therefore pleased to see a recent paper reporting tests of an siRNA-delivery system in monkeys (J. Heidel et al. Proc. Natl Acad. Sci. USA 104, 5715–5721; 2007), suggesting that safe, repeated systemic administration of siRNAs is possible.

Mark Davis of the California Institute of Technology in Pasadena and his colleagues created nanoparticles composed of siRNAs and a novel polymer based on the sugar cyclodextrin. These particles were injected into the monkeys and their health was monitored. The monkeys tolerated multiple doses of siRNA of increasing amounts.

This paper was of interest to me not only because my group works on lipid formations that might serve as delivery systems for siRNA or other genetic agents, but also because I was pleased to see a former student doing well. Jeremy, the first author, once worked in my lab as an undergraduate.

Studies such as this one are bringing back to the field the excitement that surrounded gene therapies in the 1980s.

Ralph Lewin

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Scripps Institution of Oceanography, La Jolla, California, USA

A marine biologist sees the potential of cyanobacteria, and the benefits of their renaming.

Let’s start with a false syllogism: bacteria are prokaryotes, blue-green algae are prokaryotes, and therefore blue-green algae are bacteria. All other algae are eukaryotes and so, the argument went, we should reclassify the Cyanophyta as cyanobacteria.

I was never in favour of this renaming, but it may have been good for funding. I’ve heard that grant applications for research on bacteria have better chances of success than those for research on blue-green algae.

And these oft-neglected organisms have a lot to offer. A recent paper on Lyngbya majuscula from Bill Gerwick, now at the Scripps Institution of Oceanography in La Jolla, California, and his colleagues (B. Han et al. J. Nat. Prod. 69, 572–575; 2006), for example, reveals some interesting new compounds.

L. majuscula grows on warm seashores as tufts, which, when they come loose and float away, can stick to swimmers’ skin and cause a rash — known as swimmers’ itch or seaweed dermatitis.

Gerwick and his team extracted from dried L. majuscula two compounds that may explain its irritant effect. The compounds, aurilide B and aurilide C, are hugely complicated ring-shaped molecules that resemble a toxin previously isolated from sea slugs.

In tissue culture assays, the compounds proved toxic to human and mouse cancer cells. Such natural products can act as starting points for pharmaceutical chemists.

Gerwick’s paper refers to L. majuscula as a cyanobacterium in its title and as an alga elswhere in its text, but what’s important is the science, not the names.