A NanoLuc luciferase-based assay enabling the real-time analysis of protein secretion and injection by bacterial type III secretion systems.

The elucidation of the molecular mechanisms of secretion through bacterial protein secretion systems is impeded by a shortage of assays to quantitatively assess secretion kinetics. Also the analysis of the biological role of these secretion systems as well as the identification of inhibitors targeting these systems would greatly benefit from the availability of a simple, quick and quantitative assay to monitor principle secretion and injection into host cells. Here, we present a versatile solution to this need, utilizing the small and very bright NanoLuc luciferase to assess the function of the type III secretion system encoded by Salmonella pathogenicity island 1. Type III secretion substrate-NanoLuc fusions are readily secreted into the culture supernatant, where they can be quantified by luminometry after removal of bacteria. The NanoLuc-based secretion assay features a very high signal-to-noise ratio and sensitivity down to the nanolitre scale. The assay enables monitoring of secretion kinetics and is adaptable to a high throughput screening format in 384-well microplates. We further developed a split NanoLuc-based assay that enables the real-time monitoring of type III secretion-dependent injection of effector-HiBiT fusions into host cells stably expressing the complementing NanoLuc-LgBiT.

Horizontal Natural Product Transfer: Intriguing Insights into a Newly Discovered Phenomenon.

Just recently, the "horizontal natural product transfer" was unveiled: alkaloids, which have been leached out from decomposing alkaloidal donor plants, are taken up by the roots of acceptor plants. In the same manner, many other natural products, such as coumarins or stilbenes, are also taken up from the soil. Recent research outlined that alkaloids are transferred also from a living donor plant to plants growing in their vicinity. In the acceptor plants, the imported natural products might be modified by hydroxylation and glucosylation. These insights will strongly impact our understanding of contamination of plant-derived commodities as well as plant-plant interactions.

Transfer of pyrrolizidine alkaloids between living plants: A disregarded source of contaminations.

To elucidate the origin of the wide-spread contaminations of plant derived commodities with various alkaloids, we employed co-cultures of pyrrolizidine alkaloid (PA) containing Senecio jacobaea plants with various alkaloid free acceptor plants. Our analyses revealed that all plants grown in the vicinity of the Senecio donor plants indeed contain significant amounts of the PAs, which previously had been synthesized in the Senecio plants. These findings illustrate that typical secondary metabolites, such as pyrrolizidine alkaloids, are commonly transferred and exchanged between living plants. In contrast to the broad spectrum of alkaloids in Senecio, in the acceptor plants nearly exclusively jacobine is accumulated. This indicates that this alkaloid is exuded specifically by the Senecio roots. Although the path of alkaloid transfer from living donor plants is not yet fully elucidated, these novel insights will extend and change our understanding of plant-plant interactions and reveal a high relevance with respect to the widespread alkaloidal contaminations of plant-derived commodities. Moreover, they could be the basis for the understanding of various so far not fully understood phenomena in cultivation of various crops, e.g. the beneficial effects of crop rotations or the co-cultivation of certain vegetables.

Uptake of nicotine from discarded cigarette butts - A so far unconsidered path of contamination of plant-derived commodities.

This study aimed to elucidate the origin of the widespread nicotine contamination of plant-derived commodities, by conducting field experiments with various herbs and spice plants. By scattering tobacco and cigarette butts on the field and subsequent nicotine analyses of the acceptor plants, we verified that the alkaloid is leached out into the soil and is taken up by the crop plants. This path of contamination pertains even when there is only one cigarette butt per square meter. Even such minor pollution results - at least in the case of basil and peppermint - in considerable high nicotine contaminations, which exceed the maximum residue level by more than 20-fold. The data reported here clearly outline the large practical relevance of this soil-borne contamination path and imply that unthoughtful disposal of cigarette butts in the field by farm workers may be the reason for the widespread occurrence of nicotine contamination in plant-derived commodities. Therefore, such misbehavior needs to be prevented using education and sensitization, and by including this issue into the guidelines of good agricultural practice.

Horizontal natural product transfer: A potential source of alkaloidal contaminants in phytopharmaceuticals.

BACKGROUND: It was recently shown that nicotine and pyrrolizidine alkaloids that leach out from decomposing plant material (donor plants) are subsequently taken up by the roots of acceptor plants and translocated into their leaves. Furthermore, it is well established that plant roots take up xenobiotics, generally by simple diffusion, and that this passive import depends on the physico-chemical properties of the substances. HYPOTHESIS: Based on the well-known uptake of xenobiotics, we assumed that in analogy, the uptake of alkaloids, which are leached out from plant material (donor plants) represents a quite general feature of plant biology. METHODS: Using barley as a model plant, we analyzed the uptake of alkaloids by applying them to Hordeum vulgare seedlings. Based on HPLC analyses, the presence of the particular alkaloids in the acceptor plants was determined. RESULTS: We demonstrated that numerous alkaloids of different structural types are able to diffuse through biomembranes and are taken up by acceptor plants. In contrast, an uptake of quaternary alkaloids, with a permanent positive charge, could not be detected. CONCLUSION: As most alkaloidal plants generally die back afield, and the corresponding natural products are leached out into the soil. Our findings have substantial relevance for all plant-derived commodities, especially for the production of phytopharmaceuticals and the related safety issues. Moreover, the evidence that plants are inherently able to take up alkaloids from the soil, which are derived from other plants, will alter our appraisal of plant-plant interactions. In this context, the classical definition of xenobiotics, which are considered as "non-natural" substances, might be also extended by including natural products leached out into the soil.

Interspecific transfer of pyrrolizidine alkaloids: An unconsidered source of contaminations of phytopharmaceuticals and plant derived commodities.

Many plant derived commodities contain traces of toxic pyrrolizidine alkaloids (PAs). The main source of these contaminations seems to be the accidental co-harvest of PA-containing weeds. Yet, based on the insights of the newly described phenomenon of the horizontal transfer of natural products, it is very likely that the PA-contaminations may also be due to an uptake of the alkaloids from the soil, previously being leached out from rotting PA-plants. The transfer of PAs was investigated using various herbs, which had been mulched with dried plant material from Senecio jacobaea. All of the acceptor plants exhibited marked concentrations of PAs. The extent and the composition of the imported PAs was dependent on the acceptor plant species. These results demonstrate that PAs indeed are leached out from dried Senecio material into the soil and confirm their uptake by the roots of the acceptor plants and the translocation into the leaves.

Stress-related femoral cortical and cancellous bone density loss after collum femoris preserving uncemented total hip arthroplasty: a prospective 7-year follow-up with quantitative computed tomography.

PURPOSE: Bone loss around uncemented femoral components is suspected to precede implant loosening and contribute to problems in revision surgery. Short-stemmed cementless femoral components are designed to preserve proximal femoral bone stock and ultimately the longevity of the prosthesis. METHODS: With quantitative computed tomography-assisted osteodensitometry, we prospectively analyzed femoral cortical and cancellous bone density (BD) and contact area changes of an uncemented collum femoris preserving stem (n = 38) 10 days, 1, 3 and 7 years post-operatively. RESULTS: Seven years post-operatively, cancellous BD (mg CaHA/mL) had decreased by as much as -66 % and cortical BD by up to -27 % at the metaphyseal portion of the femur; the decrease was progressive between the 1- and 3-year examinations and halted thereafter. Contact area (in % out of a possible 100 %) decreased proximally between the 1- and 3-year follow-up. CONCLUSION: Proximal cortical and cancellous bone density loss and decrease of contact area indicate that metaphyseal fixation cannot be achieved. The lack of cortical BD loss and increase of contact area values below the trochanter minor suggest diaphyseal fixation.

Periacetabular cortical and cancellous bone mineral density loss after press-fit cup fixation: a prospective 7-year follow-up.

The impact of total hip arthroplasty on strain adaptive bone remodeling has been extensively analyzed by dual-energy x-ray absorptiometry. In this study, we present a prospective computed tomography-assisted study of periacetabular cortical and cancellous bone mineral density (in milligrams of calcium hydroxyapatite [CaHA] per milliliter, or mgCaHA/mL) changes 10 days and 1, 3, and 7 years after press-fit cup implantation for 38 hips in vivo. Cancellous bone mineral density decreased by Ø -63% ventral and Ø -85% dorsal to the cup; cortical bone mineral density, by Ø -22% ventral and Ø -18% dorsal to the cup. The presented periacetabular strain adaptive bone mineral density data are the most extensive of the current literature. Even the measured extensive cancellous bone mineral density loss was thus far of no clinical relevance because all cups showed radiographic signs of stable ingrowth.

Prospective study of a cementless total hip arthroplasty with a collum femoris preserving stem and a trabeculae oriented pressfit cup: minimun 6-year follow-up.

AIM: Tissue sparing hip prostheses are becoming more and more popular especially for the treatment of younger patients. The objective of this study was to evaluate the clinical and radiological results after insertion of a tissue sparing hip prosthesis. METHOD: In 47 consecutive non-selected patients (50 hips), the clinical and radiographic results of cementless total hip arthroplasty using a collum femoris preserving stem, a pressfit cup and an alumina-polyethylene pairing were prospectively evaluated. The mean age at index-surgery was 58 (36-82) years, there were 38 women and 9 men. After a mean follow-up of 6.8 years (minimum 6.1 years), two patients had died and one patient underwent revision surgery due to aseptic loosening of the stem. RESULTS: The overall survival rate of the acetabular component was 100% and of the femoral component 98%. The mean Harris hip score at follow-up was 94 points. CONCLUSION: The mid-term survival with this type of total hip arthroplasty is excellent and compares equal with uncemented straight stems in this age group.