Small angle X-ray scattering as a high-throughput method to classify antimicrobial modes of action.

Multi-drug resistant bacteria are currently undermining our health care system worldwide. While novel antimicrobial drugs, such as antimicrobial peptides, are urgently needed, identification of new modes of action is money and time consuming, and in addition current approaches are not available in a high throughput manner. Here we explore how small angle X-ray scattering (SAXS) as high throughput method can contribute to classify the mode of action for novel antimicrobials and therefore supports fast decision making in drug development. Using data bases for natural occurring antimicrobial peptides or predicting novel artificial peptides, many candidates can be discovered that will kill a selected target bacterium. However, in order to narrow down the selection it is important to know if these peptides follow all the same mode of action. In addition, the mode of action should be different from conventional antibiotics, in consequence peptide candidates can be developed further into drugs against multi-drug resistant bacteria. Here we used one short antimicrobial peptide with unknown mode of action and compared the ultrastructural changes of Escherichia coli cells after treatment with the peptide to cells treated with classic antibiotics. The key finding is that SAXS as a structure sensitive tool provides a rapid feedback on drug induced ultrastructural alterations in whole E. coli cells. We could demonstrate that ultrastructural changes depend on the used antibiotics and their specific mode of action. This is demonstrated using several well characterized antimicrobial compounds and the analysis of resulting SAXS curves by principal component analysis. To understand the result of the PCA analysis, the data is correlated with TEM images. In contrast to real space imaging techniques, SAXS allows to obtain nanoscale information averaged over approximately one million cells. The measurement takes only seconds, while conventional tests to identify a mode of action require days or weeks per single substance. The antimicrobial peptide showed a different mode of action as all tested antibiotics including polymyxin B and is therefore a good candidate for further drug development. We envision SAXS to become a useful tool within the high-throughput screening pipeline of modern drug discovery. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

Effects of a Brown-midrib corn hybrid on nutrient digestibility in wethers and on dry matter intake, performance, rumen and blood variables in dairy cows.

The aim of the present trials was to determine the effect of an experimental Brown-midrib (Bm) corn hybrid in relation to a commercial corn hybrid (Con) on digestibility in wethers and on dry matter intake (DMI), milk yield and milk composition in dairy cows. Digestibility of crude fibre (CF), neutral detergent fibre (NDFom) and acid detergent fibre (ADFom) were higher for Bm (CF Con: 57.8%; Bm: 67.2%; NDFom Con: 56.8%; Bm: 64.8%; ADFom Con: 52.0%; Bm: 63.9%), but concentration of net energy for lactation did not differ (Con: 6.4 MJ/kg DM; Bm: 6.3 MJ/kg DM). A total of 64 lactating German Holstein cows were assigned to one of the two dietary treatments Con or Bm according to milk yield, lactation number, days in milk and live weight. In Trial 1, cows were fed a total mixed ration consisting of 50% corn silage (Con or Bm) and 50% concentrate on dry matter (DM) basis. In Trial 2, the same animals were fed the respective silage for ad libitum intake and 5.3 kg of concentrate DM per animal per day. In Trial 1, DMI and milk-fat content were decreased significantly for the Bm-treatment (DMI Con: 22.5 kg/day; Bm: 21.5 kg/day; milk fat Con: 3.8%; Bm: 3.3%). In Trial 2, milk yield and fat-corrected milk (FCM) were increased significantly, whereas milk-fat% was decreased significantly (milk yield Con: 25.8 kg/day; Bm: 29.4 kg/day; FCM Con: 27.2 kg/day; Bm: 29.6 kg/day; fat Con: 4.4%; Bm: 4.0%). Diets did not influence ruminal pH or temperature. Diets, furthermore, did not influence rumination in either trial. Additional research on digestibility and rumen fermentation should, however, be carried out using dairy cows at respective intake levels as trials with wethers cannot be transferred to high-yielding ad libitum fed cows.

Single pulse coherence measurements in the water window at the free-electron laser FLASH.

The spatial coherence of free-electron laser radiation in the water window spectral range was studied, using the third harmonic (λ<(3rd) = 2.66 nm) of DESY's Free-electron LASer in Hamburg (FLASH). Coherent single pulse diffraction patterns of 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) multilamellar lipid stacks have been recorded. The intensity histogram of the speckle pattern around the first lamellar Bragg peak, corresponding to the d = 5 nm periodicity of the stack, reveals an average number of transverse modes of M¯ = 3.0 of the 3rd harmonic. Using the lipid stack as a 'monochromator', pulse-to-pulse fluctuations in the third harmonic λ(3rd) have been determined to be 0.033 nm.

Spatial and temporal coherence properties of single free-electron laser pulses.

The experimental characterization of the spatial and temporal coherence properties of the free-electron laser in Hamburg (FLASH) at a wavelength of 8.0 nm is presented. Double pinhole diffraction patterns of single femtosecond pulses focused to a size of about 10×10 µm(2) were measured. A transverse coherence length of 6.2 ± 0.9 µm in the horizontal and 8.7 ± 1.0 µm in the vertical direction was determined from the most coherent pulses. Using a split and delay unit the coherence time of the pulses produced in the same operation conditions of FLASH was measured to be 1.75 ± 0.01 fs. From our experiment we estimated the degeneracy parameter of the FLASH beam to be on the order of 10(10) to 10(11), which exceeds the values of this parameter at any other source in the same energy range by many orders of magnitude.

Digital in-line X-ray holography with zone plates.

Single pulse imaging with radiation provided by free-electron laser sources is a promising approach towards X-ray microscopy, which is expected to provide high resolution images of biological samples unaffected by radiation damage. One fully coherent imaging technique for this purpose is digital in-line holography. Key to its successful application is the creation of X-ray point sources with high photon flux. In this study we applied zone plates to create such point sources with synchrotron radiation provided by the storage ring BESSY II. The obtained, divergent light cone is applied to holographic microscopy of biological objects such as critical point dried Navicula perminuta diatoms and human cells using photons with an energy of 250 eV. Compared to conventional experiments employing pinholes, exposure times are reduced by two orders of magnitude.

X-ray holographic microscopy with zone plates applied to biological samples in the water window using 3rd harmonic radiation from the free-electron laser FLASH.

The imaging of hydrated biological samples - especially in the energy window of 284-540 eV, where water does not obscure the signal of soft organic matter and biologically relevant elements - is of tremendous interest for life sciences. Free-electron lasers can provide highly intense and coherent pulses, which allow single pulse imaging to overcome resolution limits set by radiation damage. One current challenge is to match both the desired energy and the intensity of the light source. We present the first images of dehydrated biological material acquired with 3rd harmonic radiation from FLASH by digital in-line zone plate holography as one step towards the vision of imaging hydrated biological material with photons in the water window. We also demonstrate the first application of ultrathin molecular sheets as suitable substrates for future free-electron laser experiments with biological samples in the form of a rat fibroblast cell and marine biofouling bacteria Cobetia marina.

Ptychographic coherent x-ray diffractive imaging in the water window.

Coherent x-ray diffractive microscopy enables full reconstruction of the complex transmission function of an isolated object to diffraction-limited resolution without relying on any optical elements between the sample and detector. In combination with ptychography, also specimens of unlimited lateral extension can be imaged. Here we report on an application of ptychographic coherent diffractive imaging (PCDI) in the soft x-ray regime, more precisely in the so-called water window of photon energies where the high scattering contrast between carbon and oxygen is well-suited to image biological samples. In particular, we have reconstructed the complex sample transmission function of a fossil diatom at a photon energy of 517 eV. In imaging a lithographically fabricated test sample a resolution on the order of 50 nm (half-period length) has been achieved. Along with this proof-of-principle for PCDI at soft x-ray wavelengths, we discuss the experimental and technical challenges which can occur especially for soft x-ray PCDI.