Massively parallel single-molecule manipulation using centrifugal force

Precise manipulation of single molecules has already led to remarkable insights in physics, chemistry, biology and medicine. However, widespread adoption of single-molecule techniques has been impeded by equipment cost and the laborious nature of making measurements one molecule at a time. We have solved these issues with a new approach: massively parallel single-molecule force measurements using centrifugal force. This approach is realized in a novel instrument that we call the Centrifuge Force Microscope (CFM), in which objects in an orbiting sample are subjected to a calibration-free, macroscopically uniform force-field while their micro-to-nanoscopic motions are observed. We demonstrate high-throughput single-molecule force spectroscopy with this technique by performing thousands of rupture experiments in parallel, characterizing force-dependent unbinding kinetics of an antibody-antigen pair in minutes rather than days. Additionally, we verify the force accuracy of the instrument by measuring the well-established DNA overstretching transition at 66 $\pm$ 3 pN. With significant benefits in efficiency, cost, simplicity, and versatility, "single-molecule centrifugation" has the potential to revolutionize single-molecule experimentation, and open access to a wider range of researchers and experimental systems.Comment: 5 pages, 3 figure

Från aspekt till övergripande – en ordlista över svensk akademisk vokabulär

This report describes a project to develop an academic word list for Swedish. The resulting word list is published at <http://spraakbanken.gu.se/ao/>. It comprises 655 headwords, extracted from a 25 million word corpus of Swedish academic texts. Both the word list and the corpus are openly accessible through Språkbanken’s lexical and corpus infrastructures

A high-resolution magnetic tweezer for single-molecule measurements

Magnetic tweezers (MT) are single-molecule manipulation instruments that utilize a magnetic field to apply force to a biomolecule-tethered magnetic bead while using optical bead tracking to measure the biomolecule’s extension. While relatively simple to set up, prior MT implementations have lacked the resolution necessary to observe sub-nanometer biomolecular configuration changes. Here, we demonstrate a reflection-interference technique for bead tracking, and show that it has much better resolution than traditional diffraction-based systems. We enhance the resolution by fabricating optical coatings on all reflecting surfaces that optimize the intensity and contrast of the interference image, and we implement feedback control of the focal position to remove drift. To test the system, we measure the length change of a DNA hairpin as it undergoes a folding/unfolding transition

Harbouring public good mutants within a pathogen population can increase both fitness and virulence

Existing theory, empirical, clinical and field research all predict that reducing the virulence of individuals within a pathogen population will reduce the overall virulence, rendering disease less severe. Here, we show that this seemingly successful disease management strategy can fail with devastating consequences for infected hosts. We deploy cooperation theory and a novel synthetic system involving the rice blast fungus Magnaporthe oryzae. In vivo infections of rice demonstrate that M. oryzae virulence is enhanced, quite paradoxically, when a public good mutant is present in a population of high-virulence pathogens. We reason that during infection, the fungus engages in multiple cooperative acts to exploit host resources. We establish a multi-trait cooperation model which suggests that the observed failure of the virulence reduction strategy is caused by the interference between different social traits. Multi-trait cooperative interactions are widespread, so we caution against the indiscriminant application of anti-virulence therapy as a disease-management strategy

Mutations Altering the Interplay between GkDnaC Helicase and DNA Reveal an Insight into Helicase Unwinding

Replicative helicases are essential molecular machines that utilize energy derived from NTP hydrolysis to move along nucleic acids and to unwind double-stranded DNA (dsDNA). Our earlier crystal structure of the hexameric helicase from Geobacillus kaustophilus HTA426 (GkDnaC) in complex with single-stranded DNA (ssDNA) suggested several key residues responsible for DNA binding that likely play a role in DNA translocation during the unwinding process. Here, we demonstrated that the unwinding activities of mutants with substitutions at these key residues in GkDnaC are 2–4-fold higher than that of wild-type protein. We also observed the faster unwinding velocities in these mutants using single-molecule experiments. A partial loss in the interaction of helicase with ssDNA leads to an enhancement in helicase efficiency, while their ATPase activities remain unchanged. In strong contrast, adding accessory proteins (DnaG or DnaI) to GkDnaC helicase alters the ATPase, unwinding efficiency and the unwinding velocity of the helicase. It suggests that the unwinding velocity of helicase could be modulated by two different pathways, the efficiency of ATP hydrolysis or protein-DNA interaction

The mechanism of DNA unwinding by the eukaryotic replicative helicase

Accurate DNA replication is tightly regulated in eukaryotes to ensure genome stability during cell division and is performed by the multi-protein replisome. At the core an AAA+ hetero-hexameric complex, Mcm2-7, together with GINS and Cdc45 form the active replicative helicase Cdc45/Mcm2-7/GINS (CMG). It is not clear how this replicative ring helicase translocates on, and unwinds, DNA. We measure real-time dynamics of purified recombinant Drosophila melanogaster CMG unwinding DNA with single-molecule magnetic tweezers. Our data demonstrates that CMG exhibits a biased random walk, not the expected unidirectional motion. Through building a kinetic model we find CMG may enter up to three paused states rather than unwinding, and should these be prevented, in vivo fork rates would be recovered in vitro. We propose a mechanism in which CMG couples ATP hydrolysis to unwinding by acting as a lazy Brownian ratchet, thus providing quantitative understanding of the central process in eukaryotic DNA replication

Step by step. A computational analysis of Swedish textbook language

In this work, I present a linguistic investigation of the language of Swedish textbooks in the natural sciences, i.e., biology, physics and chemistry. The textbooks, which are used in secondary and upper secondary school, are examined with respect to traditional readability measures, e.g., LIX, OVIX and nominal ratio. I also extract typical linguistic features of the texts, typicality being determined using a proposed quantitative method, labelled the index principle. This empirical, corpus-based method relies on automatic linguistic annotations produced by language technology tools to calculate what I call index lists, rank-ordered lists of characteristic linguistic features of specific text corpora as compared to reference texts. I produce index lists for typical vocabulary, noun phrase structures and syntactic structures, extracted from a 5.2 million word textbook corpus, compiled as a part of the work presented. As well as being frequent and well dispersed, the linguistic variables selected for the index lists are also characteristic of the text type in question, as is evident when they are compared to a reference corpus, comprising textbooks in the social sciences and mathematics, as well as narrative and academic (university-level) texts. The results show that textbooks in natural science contain a lot of content-specific, technical vocabulary. This characteristic not only distinguishes natural scientific language from everyday language, but also from social scientific language, which on the lexical level has more in common with narrative texts. On the other hand, the textbook language as a whole is structurally distinguishable from narrative texts, as clearly seen, e.g., in its noun phrase complexity. In the transition between secondary and upper secondary school, the scores of almost every readability measure go up, indicating an increase in linguistic demands on the readers. In the upper secondary textbooks the words are longer, the vocabulary more varied, the noun phrases longer and more elaborate, and the most typical syntactic structures more complex. Notably, the linguistic development between the form levels is more marked in the natural-science textbooks, compared to social sciences and mathematics. Nevertheless, the textbook language overall shows a relatively low complexity in comparison to academic language