Patricia Turner

Nitrogen fixation: Nitrogenase genes and gene expression

Publisher Summary This chapter discusses nitrogenase genes and gene expressions. The primers used for nifH amplification, the methods used to extract genomic DNA and mRNA, the alignment and analysis of nifH sequences, and the RT-PCR protocol are described. Biological nitrogen fixation is the enzymatic reduction of atmospheric dinitrogen to ammonium. The conventional nitrogenase enzyme is encoded by the nifHDK genes, which are in contiguous arrangement within the genome. Alternative nitrogenases (alternative and second alternative) also contain nifH, but contain a third protein in the counterpart to the Mo protein, which is encoded by nifG (nifDGK). Nitrogenase genes can be detected and characterized by amplification from environmental samples using the polymerase chain reaction (PCR). Amplification of nitrogenase genes indicates that nitrogen-fixing microorganisms are present, but not whether or not they are actively fixing nitrogen. By coupling, the PCR assay with reverse transcription (RT-PCR) microorganisms that are actively expressing the nitrogenase enzyme can be detected. Once genes are amplified, the diversity of sequences can be determined by a number of means, including cloning and sequencing of individual amplification products.

The bone diagnostic instrument II: Indentation distance increase

The bone diagnostic instrument (BDI) is being developed with the long-term goal of providing a way for researchers and clinicians to measure bone material properties of human bone in vivo. Such measurements could contribute to the overall assessment of bone fragility in the future. Here, we describe an improved BDI, the Osteoprobe IItrade mark. In the Osteoprobe IItrade mark, the probe assembly, which is designed to penetrate soft tissue, consists of a reference probe (a 22 gauge hypodermic needle) and a test probe (a small diameter, sharpened rod) which slides through the inside of the reference probe. The probe assembly is inserted through the skin to rest on the bone. The distance that the test probe is indented into the bone can be measured relative to the position of the reference probe. At this stage of development, the indentation distance increase (IDI) with repeated cycling to a fixed force appears to best distinguish bone that is more easily fractured from bone that is less easily fractured. Specifically, in three model systems, in which previous mechanical testing and/or tests reported here found degraded mechanical properties such as toughness and postyield strain, the BDI found increased IDI. However, it must be emphasized that, at this time, neither the IDI nor any other mechanical measurement by any technique has been shown clinically to correlate with fracture risk. Further, we do not yet understand the mechanism responsible for determining IDI beyond noting that it is a measure of the continuing damage that results from repeated loading. As such, it is more a measure of plasticity than elasticity in the bone.

Bone diagnostic instrument

The bone diagnostic instrument is designed to measure materials properties of bone even if it is covered with soft tissue such as periosteum, connective tissue and skin. It uses (1) a probe assembly, consisting of a reference probe that penetrates soft tissue and stops on the surface of the bone and a test probe that is inserted into the bone, (2) an actuation system that can move the test probe, typically into and out of the bone, (3) a sensing system that can determine the dynamics of the test probe as it moves in the bone, and (4) a measurement system to record the data that is sensed during the motion. In our current prototype, a sharpened, solid test probe slides inside a sharpened hypodermic syringe that serves as the reference probe. A load cell senses the force as a function of the distance that the test probe is inserted into the bone relative to the position of the reference probe that rests on the surface of the bone, measured with a linear variable displacement transformer. Examples of the type of data that can be taken with this prototype include cyclic force versus distance curves that show differences in material properties of different types of bone. (c) 2006 American Institute of Physics.

Outstanding meeting paper: high-speed photography of the development of microdamage in trabecular bone during compression

The mechanical properties of healthy and diseased bone tissue are extensively studied in mechanical tests. Most of this research is motivated by the immense costs of health care and social impacts due to osteoporosis in post-menopausal women and the aged. Osteoporosis results in bone loss and change of trabecular architecture, causing a decrease in bone strength. To address the problem of assessing local failure behavior of bone, we combined mechanical compression testing of trabecular bone samples with high-speed photography. In this exploratory study, we investigated healthy, osteoarthritic, and osteoporotic human vertebral trabecular bone compressed at high strain rates. Apparent strains were found to transfer into to a broad range of local strains. Strained trabeculae were seen to whiten with increasing strain. Comparison of whitened regions seen in high-speed photography sequences with scanning electron micrographs showed that the observed whitening was due to the formation of microcracks. From the results of a motion energy filter applied to the recorded movies, we saw that the whitened areas are, presumably, also areas of high deformation. In summary, high-speed photography allows the detection of microdamage in real time, leading toward a better understanding of the local processes involved in bone failure.

Publisher’s Note: “The bone diagnostic instrument II: Indentation distance increase” [Rev. Sci Instrum. 79, 064303 (2008)]

Note: Times Cited: 0 Reference EPFL-ARTICLE-154171doi:10.1063/1.3142471View record in Web of Science Record created on 2010-11-05, modified on 2017-12-17

The bone diagnostic instrument II: Indentation distance increase (vol 79, 064303, 2008)

Note: Times Cited: 0 Reference EPFL-ARTICLE-154171doi:10.1063/1.3142471View record in Web of Science Record created on 2010-11-05, modified on 2017-12-17

Publisher’s Note: “The bone diagnostic instrument II: Indentation distance increase”.

Note: Times Cited: 0 Reference EPFL-ARTICLE-154171doi:10.1063/1.3142471View record in Web of Science Record created on 2010-11-05, modified on 2017-12-17