Therefore, it is currently widely used in studies on clinical diagnosis of Parkinson’s disease (PD) and movement-related disorders. In conventional 99mTc-TRODAT-1 SPECT image evaluation, visual inspection or manual selection of ROI for semiquantitative analysis is mainly used to observe and evaluate the degree of striatal defects. However, these methods are dependent on the subjective opinions of observers, which lead to human errors, have shortcomings such as long duration, increased effort, and have low reproducibility. To solve this problem, this study aimed to establish an automatic semiquantitative analytical method for 99mTc-TRODAT-1. This method combines three drug templates (one built-in SPECT template in SPM software and two self-generated MRI-based and HMPAO-based TRODAT-1 templates) for the semiquantitative analysis of the striatal phantom and clinical images. At the same time, the results of automatic analysis of the three templates were compared with results from a conventional manual analysis for examining the feasibility of automatic analysis and the effects of drug templates on automatic semiquantitative analysis results. After comparison, it was found that the MRI-based TRODAT-1 template generated from MRI images is the most suitable template for 99mTc-TRODAT-1 automatic semiquantitative analysis. This radiopharmaceutical or drug has been confirmed to bind to dopamine transporters in the living organisms [Dopamine transporters are located in the presynaptic cell membrane at dopaminergic neuronal endings and play an important role in regulating extracellular dopamine levels. Therefore, these transporters can affect the number of neural signals received by the striatum. Conditions affecting these transporters can thus cause Parkinson’s disease (PD) of varying severity. Thus, dopamine transporters could be used as a biomarker for evaluating striatal neuronal function , 2. Whenrganisms . Thereforganisms , 5.123I-FP-CIT SPECT imaging. This software provides three semiquantitative analytical methods, including the two-box method proposed by Fleming and Bolt et al. [123I-FP-CIT SPECT images for semiquantitative analysis. The study by Morton et al. compared these three methods in detail [123I-IBZM SPECT template that conforms to the Montreal Neurological Institute space (MNI space) as well as predefined ROI that similarly conforms to MNI space, and is different from the abovementioned methods. This is because all images must first undergo spatial normalization with the template, after which ROI is applied to all normalized images for semiquantitative analysis. Therefore, the drug templates play an important role in the accuracy of this analytical method.In conventional SPECT image evaluation, visual inspection or manual selection of ROI for semiquantitative analysis is mainly used to evaluate the degree of striatal defects . Howevert et al. , the thrt et al. , and thet et al. . These tn detail , 12. Apan detail  using st99mTc-TRODAT-1. Among these templates, two were 99mTc-TRODAT-1 templates created in this study, whereas another was the SPECT template that is built-in in SPM. The three templates are all used in striatal phantoms and clinical images for semiquantitative analysis. At the same time, the results of semiquantitative analysis using these three methods were compared with the results from conventional manual analysis to identify the optimal 99mTc-TRODAT-1 template. We hope that this template can be used in automatic semiquantitative analysis of 99mTc-TRODAT-1 SPECT images to quantify the specific uptake ratio (SUR) in images of patients with PD. In addition, we hope that this template could improve the problems associated with conventional manual selection methods such as long duration, increased effort, low reproducibility, and human subjectivity.In this study, we compared three drug templates with the aim of establishing an automatic semiquantitative analytical method exclusively for 99mTc-TRODAT-1 by intravenous injection and imaging was performed approximately 4 hours after injection [99mTc energy peaks of 140 keV ± 10% were used, and the step-and-shoot scan mode was used with 360° rotation in the clockwise direction, obtaining projections at intervals of 3°. Each projection data was collected for 20 seconds, for a total of 120 projections. The image reconstruction matrix size was 128 × 128, with every pixel size and slice thickness being 3.89 mm. The Metz filter was used for filtered backprojection (FBP) reconstruction; the cutoff frequency used for the filter was 0.55 times the Nyquist frequency and the power order was 30. First-order Chang’s method was used for attenuation correction and the attenuation coefficient (μ) was 0.12 cm−1 [This study collected images from a total of 142 subjects, including 58 healthy subjects  and 84 PD patients . The fan-beam collimator in E.CAM  was used for imaging. Every patient was administered 740 MBq (20 mCi) of njection . During 99mTc-TRODAT-1 drug for imaging of dopamine transporters. The TRODAT-1 drug was obtained from the lyophilized vials of dopamine transporter imaging reagents from the Institute of Nuclear Energy Research at Lungtan, Taiwan. Five milliliters of physiological saline containing 1480 MBq of 99mTc sodium pertechnetate (NaTcO4) was injected into the lyophilized vials and placed in a lead container for shaking until the contents have completely dissolved. The container was then placed in a high-temperature steam sterilizer and heated for 30 min at 121°C. After cooling to room temperature, the radioactivity and chemical purity were confirmed to be 99% [This experiment primarily used the 99mTc-TRODAT-1 images with the greatest activity uptake were selected and summed to form an image. ROI of the striatum and occipital lobe were manually selected on this image by experienced radiology technicians [Three transverse slices of the reconstructed hnicians , and the99mTc-TRODAT-1 images. The “Normalise” module of the SPM8 software (http://www.fil.ion.ucl.ac.uk/spm/software/) was used in our custom-made software [99mTc-TRODAT-1 images was first performed using a standard MNI space brain template. The minimized mean-squared difference between the source image and drug template was calculated so that the spatial coordinates of the source image and the size of voxels were consistent with the template in standard MNI space [3. Finally, the region of caudate, putamen, and occipital lobe defined in the automated anatomical labeling (AAL) ROI template that also conformed to MNI spatial coordinate axes was used for automatic ROI selection of normalized 99mTc-TRODAT-1 images , and it could perform analysis and evaluation of software , 16. SpaNI space , 17, 18.1 images . Similar99mTc-HMPAO cerebral blood perfusion template. Because the distribution and pharmacokinetic characteristics of 99mTc-HMPAO show large differences with those of 99mTc-TRODAT-1, this will inevitably affect the accuracy of subsequent semiquantitative analysis; therefore, a template that is exclusive for 99mTc-TRODAT-1 is considerably important. However, there is no template that is exclusive to 99mTc-TRODAT-1 currently. Therefore, before performing automatic analysis, we must first use normal 99mTc-TRODAT-1 images to create study specific templates. There are many methods for generating templates. In this study, two different templates were first generated according to different methods, which were the HMPAO-based TRODAT (HBT) template that uses the built-in 99mTc-HMPAO template in SPM as the basis and the MRI-based TRODAT (MBT) template that uses MRI images as the basis [99mTc-HMPAO provided in the publicly available SPM8 suite for accuracy comparison . All 99mTc-TRODAT-1 images that had undergone spatial normalization were then averaged and the images obtained at this point are preliminary HBT (pHBT) templates. Afterward, to increase and ensure the accuracy of spatial alignment, the above actions were repeated and all 99mTc-TRODAT-1 images underwent spatial normalization again, but the normalization template was changed to pHBT . Finally, the 99mTc-TRODAT-1 images that had undergone spatial normalization were averaged and an isotropic Gaussian kernel smoothing with 8-mm full-width-at-half-maximum (FWHM) was used [The was used , and thewas used , 20.99mTc-TRODAT-1 SPECT imaging. First, the default settings in the “Coregister: Estimate” module in SPM8 was used for the registration of the MRI T1 images and 99mTc-TRODAT-1 images of these 7 healthy subjects so that the spatial anatomical coordinates of the two conform to each other . Following that, the “Normalise: Estimate” module in SPM8 was used for spatial normalization of the MRI T1 images of the subjects using the MRI T1 template that was built-in in SPM8 . The normalization parameters were the same as those mentioned above. Then, the transformation parameters (*_sn.mat) produced from the “Normalise: Estimate” module was written into the corresponding registered 99mTc-TRODAT-1 image by using “Normalise: Write” module . All registered 99mTc-TRODAT-1 images were averaged after this indirect spatial normalization method. Finally, an 8-mm FWHM Gaussian kernel smoothing was performed [Among all the healthy subjects, 7 subjects underwent both MRI T1-weighted and erformed , 10, anderformed .Target represents the average value of the target region ROI (striatum), and Background represents the average value of the background region ROI .SUR is a commonly used marker or figure of merit (FOM) for semiquantitative analysis in brain nuclear medicine imaging . Through99mTc-TRODAT-1 images of patients .After all clinical iability  and intriability , 22. The99mTc-TRODAT-1 SUR maps of different transverse slices when different templates were used for automatic ROI selection. We can see that the MBT template has the highest positional accuracy for AAL automatic ROI selection, followed by the HBT template; the HMPAO template had the worst accuracy. 99mTc-TRODAT-1 automatic ROI selection and semiquantitative analysis, the selection of appropriate drug template is considerably important for the results of semiquantitative analysis. This is because the template will affect the accuracy of the standard MNI spatial location of the image after normalization, thereby affecting the accuracy of AAL ROI selection. Therefore, the use of an appropriate drug template can effectively increase the reliability and accuracy of semiquantitative data. Fang et al. improved the reliability and accuracy of an automatic semiquantitative analysis [99mTc-TRODAT-1 drug templates, whereas another was the HMPAO SPECT template that was built-in in SPM. We also examined and compared the effects of these three templates on semiquantitative analysis of phantom and clinical images.With regard to analysis . They prWith regard to phantom images, the SUR values calculated using the MBT template has the smallest percent error (< 2%) when compared with the SUR value calculated using conventional manual ROI analysis. Therefore, with regard to automatic analysis, combination with MBT template has some feasibility and can effectively replace conventional manual analysis and improve the shortcomings associated with conventional methods, such as long duration, increased effort, low reproducibility, and human subjectivity. In the meantime, we have measured the time of automatic analysis. In all cases the analyses were performed on a 3.4 GHz Intel (R) Core (TM) i7 64 bit CPU, and the computation and analysis time per subject was about 10 seconds when using our software. This really indicates that the method can successfully save lots of time when compared with conventional method. It is worth noting that the SUR value calculated using the HMPAO template showed a phenomenon of being negative value. This shows that the activity of the target region that is selected by AAL ROI is lower than the activity of the background region. The reason for this is that the radiotracer uptake in the HMPAO template is different from that in the image to be analyzed, resulting in errors being produced during the process of spatial normalization of image intensity and causing inaccurate anatomical locations in the standard MNI space in the image after normalization. This causes the AAL ROI to select wrong locations, thereby resulting in erroneous results. This result once again demonstrated that the use of inappropriate drug templates has a considerable effect on the results of semiquantitative analysis. With regard to accuracy of spatial alignment, we performed two spatial normalizations to generate a HBT template and found that the accuracy of the analysis results was greatly improved as percent error was decreased from 109.09% to 4.80%. Although accuracy was significantly improved, this still cannot be compared with the results of the MBT template created from MRI anatomical information.In clinical images, comparison with the results of conventional manual analysis found that the SUR value from MBT template analysis and calculation is the best in various indicators and this is consistent with the results from the phantoms. In correlation evaluations, the values calculated from MBT template had the highest correlation with conventional manual analysis results, and this approached strong correlation levels. Although HBT template results can effectively increase the accuracy of quantitative analysis and increase the correlation with manual analysis, the results are still not comparable to that of the MBT template. Therefore, the MBT template is the most suitable template among the three drug templates for use in automatic semiquantitative analysis. With regard to variability analysis, we similarly observed that the variability of the MBT template results is the lowest among the three templates. In addition, Student’s 2-sample t-tests were done  to see if there was any significant difference in variability. We found that both of the results showed a significant difference  under significant level equals to 0.05. In the Bland–Altman difference plot, the mean difference of the MBT template was the smallest among the three templates. The reason for this phenomenon is that the drug template and images that underwent spatial normalization with the MBT template has the most accurate anatomical locations in the standard MNI space. From 99mTc-TRODAT-1 images after spatial normalization using the HMPAO template and pHBT template; therefore, it still retains the distribution characteristics of its 99mTc-TRODAT-1 radiotracer and the accuracy of the semiquantitative analysis results will be better than the HMPAO template. However, its results were still not better than the results when the MBT template was used. We observed that when the HBT template was generated using the HMPAO template as the basis, the anatomical constructs in the standard MNI space will be smaller than the AAL ROI template  not the scalp. This results in smaller anatomical locations in the standard MNI space in the HBT template, and it affects the accuracy of quantitative analysis. Therefore, for construction of 99mTc-TRODAT-1 specific templates, the use of MRI images is a better method. This is because MRI provides relatively more spatial anatomical information, enabling normalized images to have more accurate anatomical locations in the standard MNI space.From the above results, the results from the use of HBT template for automatic analysis are always better than that of from the HMPAO template, regardless of whether phantom images or clinical images are used. This is because the HBT template is obtained from the averaging and blurring of template . Therefo99mTc-TRODAT-1 template, particular attention should be required in the interpretation of target region after spatial normalization. In addition, because the MBT template is created from healthy subjects, this may affect the quantitation results for PD patients with different disease severity. However, we think it is not necessary to generate disease-severity-specific templates. Even patients with the same severity, the activity distribution in the image may be different. Therefore, disease-severity-specific templates may be impractical and very hard to be generated. We believe that there still exist some relations in the quantitation results between patients with different severity when using our tracer-specific template (MBT template). As a result, we still can use this automatic quantitation method with MBT template to generate disease severity databank for diagnosis and comparison.Although MBT template is the optimal template in the automatic method, there are also some restrictions. In cases of severe brain atrophy, even when the image is matched to a specific 99mTc-TRODAT-1 drug exclusive template. The exclusive drug template established was verified for use in automatic ROI selection for semiquantitative analysis of 99mTc-TRODAT-1 SPECT images to quantify SUR of PD images, and this could improve the problems associated with conventional manual selection methods such as long duration, increased effort, low reproducibility, and human subjectivity. In addition, because there are few patients with both 99mTc-TRODAT-1 SPECT and MRI images and even some subjects who were unable to undergo MRI imaging, therefore the use of our ready-to-use MBT templates under these circumstances could improve accuracy in semiquantitative analyses. For other agencies that wish to self-generate their own radiotracer templates, this MBT template method can be used as a basis for reference to facilitate the construction of radiotracer templates.This study compared three different templates  that were combined with automatic analysis and manual analysis. Our results showed that the MBT template is the optimal S1 Table(XLSX)Click here for additional data file.


Development and application of chemical probes to globally map key virulence proteins of pathogenic bacteria. Pseudomonas aeruginosa secretes 2-heptyl-3-hydroxy-4-quinolone (PQS), a quorum sensing (QS) signal that regulates the expression of numerous virulence genes. Here we report the development and application of chemical probes to globally map quinolone binding proteins. The revealed quinolone interactome contains both known as well as newly identified virulence factors and presents new targets for the treatment of bacterial infections.The opportunistic human pathogen  This involves the synthesis, secretion, and sensing of small signalling molecules – termed autoinducers – which activate the expression of a myriad of genes that are essential for an efficient colonization of their host environment.The emergence of Multi-Drug-Resistant (MDR) bacteria increasingly forms a major and global public health issue. Antibiotic resistance alone is predicted to kill 10 million people by 2050.Pseudomonas aeruginosa is an opportunistic human pathogen that is known to be responsible for a large variety of serious and often life-threatening diseases. Its pathogenicity is strongly related to the production of an unusually large number of virulence factors, which cause tissue damage, delayed airway epithelium wound repair, highly efficient biofilm formation and suppressed innate immune responses.H)-quinolone  and its direct precursor 2-heptyl-4(1H)-quinolone (HHQ)  .12 This i.e. 4-iodo-1-(trimethylsilyl)-1-butyne). We therefore decided to synthesize analogues with a longer side chain (8 carbons) since this modification has been shown to retain its biological activity, and more importantly, it allowed us to use a more stable intermediate (5-iodo-1-(trimethylsilyl)-1-pentyne). Both diazirine-alkyne PQS and HHQ probes were synthesized by using modified methods of diazirine and quinolone synthesis, with some clear distinctions  and formic acid (HCO2H), using microwave irradiation to provide PQS probe 1 . Encouragingly, the activities of both probes proved to be almost identical to their cognate ligands  was observed in the presence of 1 . In contt Fig. S3. As mentP. aeruginosa cultures in order to identify proteins that specifically interact with our probes. First, photolabeled proteins were biotinylated using CuAAC reactions between the alkyne moiety on the probe and biotin azide, followed by enrichment of samples via streptavidin-based affinity chromatography. Then, the pulled down proteins were on-bead digested with trypsin and the resulting tryptic peptide mixture was analyzed by liquid chromatography-mass spectrometry. This strategy has been used successfully in recent years to isolate and identify very low abundant proteins from complex proteomes.10 > 1, for 1versus the DMSO negative control were designated as true probe interactors. iBAQ intensities have been proven to provide an accurate proxy for protein abundance.i.e. proteins that have affinity for the probe, linker, resin or are secondary protein binders but are not ‘true’ PQS or HHQ binders). Interestingly, we also did not observe PqsR enrichment, but could be expected given the fact that at high PQS concentrations the expression of this receptor is strongly down-regulated.After further optimizing labeling conditions, we performed liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses in wild-type PQS is known to regulate the expression of a set of 182 genes from which a significant number are PqsR-independent.Our two probes (1 and 2) also enabled us to compare the relative proteome binding profiles PQS and HHQ. Unexpectedly, in disagreement with a previous transcriptomic study where only 3 genes were shown to be controlled to a significant degree by HHQ,P. aeruginosa. Interestingly, we identified all the proteins from the former article, with the exception of the RhrR transcriptional regulator and the protease Pfpl, suggesting that there is a significant overlap in the proteome coverage between the different probes. However, there are some key differences, such as the number of proteins identified in our study, 182, against 11 in the former one. These differences can most likely be attributed to the alternative probe designs. Indeed, differences in labelling using diazirine and azide photocrosslinkers have previously been reported.In the course of preparation of this manuscript a valuable similar study was published by Spring and coworkers,We have identified both known and previously unknown protein targets of PQS and HHQ in living bacterial cells using a chemical proteomic strategy that utilizes affinity-based photo-cross-linking probes. Our results demonstrate that our probes mimic the activity of their parent compounds using a well-established quinolone reporter strain. One of the most interesting finding to emerge from our studies is the remarkable promiscuity displayed by both PQS and HHQ to affect myriad pathways, some of which are known to play a key role in the pathogenicity and adaptability of this ubiquitous organism. Projecting forward, it will be highly valuable to experimentally validate the major quinolone interacting proteins unveiled here and to apply this methodology to unravel the mechanism of action of these molecules in other biological systems that use quinolone signaling to their benefit.There are no conflicts to declare.Supplementary informationClick here for additional data file.Supplementary informationClick here for additional data file.Supplementary informationClick here for additional data file.Supplementary informationClick here for additional data file.


In cancer, vulnerable breast epithelium malignance tendency correlates with number and activation of ErbB receptor tyrosine kinases. In the presented work, we observe ErbB receptors activated by irradiation-induced DNA injury or neuregulin- The presented matter focuses on ErbB receptor protein molecules, either as isolated residues in the cell plasma membrane or in the form of dimers and oligomeres spanning the plasma membrane or being packed into intracellular carriers. Further described are connexin-43 transmembrane proteins, as constituents of the plasma membrane-residing connexons and gap junctions. After stimulation of ErbB receptors by ionizing irradiation or neuregulin-1 (NRG-1) application or receptor attenuation by application of the therapeutic antibody trastuzumab , a combined microscopy study using confocal laser scanning microscopy (CLSM) and localization microscopy (LM) reveals the dynamics of protein accumulation and trafficking.The mamma carcinoma is the predominant form of cancer in females in postindustrial societies. The individual lifetime incidence probability ranges at 13%, with a sub-ratio of 5%–15% of these cases being males  and in bThe mentioned packing and mobilization activity can function as a tag for malignant transition in breast epithelium. Several parameters of both functional and structural character influence ErbB dimer receptor tyrosine kinases (RTK) activity. During the onset of malignancy, cells of the mammal epithelium convert their cell homeostatic character into proliferation activity . The so-Disturbed receptor protein turnover by insufficient retrograde receptor trafficking increases the number of ErbB receptors in genetically-normal cells . The actThe mobilization process of activated ErbB receptors towards their nuclear region of activity is a process that is easy to follow by conventional microscopy. Besides a direct activation of ErbB receptors through the extracellular dimerizing peptide neuregulin-1β ,16, DNA A repair ,18. Here cytosol . VesicleDouble-strand breaks in the densely-packed DNA nucleosome complexes locally release the broken DNA strands from their nucleosomal carriers ,21. The y target . HSP90α DNA-PKs) ,32. In aIn addition to the described canonical feedback loops between injured nuclear DNA and ErbB receptor dimers, a side activity of chaperones and actin-related proteins affects gap junction turnover. Gap junctions are tetrameric transmembrane protein complexes organized in groups with sixfold symmetry called connexons and arranged in crystal-approximating 2D arrays. Two opposing arrays in neighboring cells face one another and constitute a field of channel hexameres called a gap junction . While bβ , and infβ ,39. Regaβ , complexβ ,42,43, aβ ,45, gap β . This flβ . Consequβ ,49 influβ Activated ErbB receptor dimers phosphorylate the downstream protein kinases PKA, PKB , and PKC ,52, and The mobilization of ErbB receptor dimers and gap junctions interfere in several ways. Nuclear DNA injury releases signaling factors like HSPs and ARPs ,57 whichransport ,59, but ransport ,61,62. Tfficking , but in fficking ,64,65 inThe coordinated physiological activity and the temporally and spatially synchronized mobilization of ErbB receptor family members and gap junction protein complexes is a key factor in carcinogenesis induced by intrinsic genetic transformations and by extrinsic mutagen factors like high energy irradiation, the epithelial-to-mesenchymal transition, or the presence of carcinogenic compounds ,67,68. FIn conclusion, it can be stated that dimerized ErbB receptors start cytosolic kinase cascades having the gap junctions’ cytosolic regulator moieties as targets, and share common features of retrograde respectively nucleograde trafficking . This isFor the analysis of the processes sketched above, a combined examination approach using confocal laser scanning microscopy (CLSM) and a microscopy method facilitating optical identification of objects in the protein assembly size regime is a promising choice ,78. The As suggested for ErbB receptors, connexin-43 protein molecules can also be found at different levels of organization. An increase of this level as accompanying the transition from connexons to gap junctions  faces a Such a combination of two different microscopy approaches accounts for the discrete and the continuous nature of ErbB receptor and gap junction residues. It takes into consideration that the ErbB receptor and connexin-43 proteins are isolated molecules a priori, but interact, and this way organize well a defined continuum de facto. However, the individual properties and restrictions of both microscopy approaches must be carefully taken into account ,87,88. TErbB receptor protein molecules and connexin-43-containing gap junction complexes are occupied by a dynamical set of accumulation, dissociation, and higher-order organization processes. During the transition between these types of ensembles, the characteristic molecule density distributions are altered dramatically.The distance data processing and diagram display can be interpreted in a simple semi-quantitative way . There aThe same data evaluation process gives different results if applied to receptor molecules organized in several clusters . Inside The repetition of image recordings at one specimen location progressively closes the gaps between the isolated fluorescence signals without superposition of neighboring signals’ point spreads. This principle does not result in a coherent image of a specimen, but either in an image point cloud with discontinuous character recapitulating the dye distribution in the specimen or in a point matrix with individual coordinate pairs, each pointing to a single fluorescence signal locality. Here, the matrix information can be used to reconstruct a pointillism image or to graphically display point-to-point distances in the form of Cartesian diagrams and histograms, in polar diagrams, or in Voronoi patterns.Among the plethora of applications using photo-cycle burst localization microscopy, there are several approaches that deal with the interaction of isolated receptor protein molecules in the plasma membrane, as opposed to receptors organized in clusters ,90,91. TReceptor clustering covers a receptor spatial distance range in the nanometer regime, ranging from some nanometers in the case of the dimers to some hundred nanometers for the cluster diameter. This indicates that even the size of the whole cluster aggregate is not accessible by confocal laser scanning microscopy (CLSM). A realistic assumption for the lateral CLSM resolution being 400–500 nm will designate any ErbB receptor pattern in the CLSM images as coming from large range organization and dynamics not related to basic clusters. Nonetheless, ErbB receptor accumulations appear as reacting to the application of neuregulin-1 (NRG-1), trastuzumab, and irradiation. Such a reaction is supported by the literature, where the stimulation of ErbB receptors with NRG-1, the attenuation of ErbB activity by the therapeutic antibodies against ErbB 2 or ErbB 3 receptors, and the impact of high-energy irradiation is reported . A displIn difference, the localization microscopy (LM)-derived localization matrix identifies receptor dynamics related to the primary receptor cluster population. The distance range between 5 and 250 nm cover the individual ErbB receptor molecules as well as the clusters.It may be stated that the combined evaluation of CLSM and LM offers the possibility to connect the long-range receptor mobility with the short-range packing density and the mid-range clustering behavior. However, the situation is further complicated by the means of cell epitope identification. The use of monoclonal antibodies against an amino acid sequence in the ErbB receptor protein facilitates the precise identification of this protein in the cell plasma membrane and the cytosol. A second serum-derived antibody carrying several fluorescent dye molecules  binds to an arbitrary amino acid (AA) sequence in the monoclonal antibody. The population of serum antibodies is heterogeneous with respect to the target epitope bound and also with respect to the locations of the dye molecules in the antibody protein. This implies that a series of spatially- and temporally-overlapping fluorescence emission bursts can originate from one primary/secondary antibody pair. At last, the primary antibody → secondary antibody → fluorescent dye molecule complex can swing over and rotate around the ErbB epitope while randomly emitting fluorescence bursts. These basic conditions of indirect immune identification must be regarded when interpreting the short-range and mid-range dynamics of ErbB receptors after stimulation and attenuation. CLSM’s typically-increased axial  discrimination capacity as compared to wide field microscopy places an optical section across these cells. As a consequence, the out of focus plasma membrane component is not shown, and the cell border appears as a polygonal ring. This makes it possible to discriminate the population of ErbB-2 receptors located in the plasma membrane  from the population in the cytosol . The mentioned ring structure is consolidated in unstimulated and trastuzumab-attenuated MCF-7 cells. The ring vanishes after NRG-1 stimulation, and the number of vesicles in the cytosol increases. It can be deduced that the stimulation by NRG-1 mobilizes the former plasma membrane-residing ErbB-2 receptor population into the cell’s interior. The mobilization process is not induced by trastuzumab application, suggesting that trastuzumab and NRG-1 act in an antagonistic fashion on the ErbB-2 receptor mobility . The effThe CLSM recording is not vulnerable against the spatial superposition of ErbB-2 receptor location and antibody association to this location, because the CLSM spatial resolution is limited to a value above the spatial size regime where the superposition process happens. This is not the case for LM imaging, and consequently, the loss of localization precision due to indirect antibody staining requires attention. In A direct comparison of unstimulated and NRG-1-stimulated ErbB-2/3 dimer clusters reveals two main properties of the transition. First, the absolute amount of plasma membrane resident ErbB-2 epitopes is decreased, as can be seen by the reduced fluorescence signal level when comparing top image and middle image. This is in accordance with the finding in the CLSM records showing the ErbB-2/3 receptors mobilized into the cytosol. The mobilized receptor fraction is lacking at the plasma membrane. This gives rise to the assumption that the activation state of a NRG-1 stimulated MCF-7 cell is not related to an increased ErbB receptor amount in the plasma membrane. Cell activation is related to receptor mobilization. Second, the plasma membrane clusters appear less dense after NRG-1 application as compared to the NRG-1-depleted situation. Again, the reduced absolute amount of ErbB-2 protein might be the reason, but it must be stated that the reduction of protein might also result in clusters of reduced size but unchanged densities. This is not the case. Cluster diameters remain unchanged, but cluster protein density decreases.After trastuzumab application, the plasma membrane-located ErbB-2 receptor situation at cell-to-cell contact regions consolidates as compared to the unstimulated case. This recapitulates the CLSM findings where a clear accumulation at the cell borders can also be seen. The LM images show the clusters being more solid than after NRG-1 stimulation, and of a size comparable to clusters in unstimulated cells. It might be deduced that trastuzumab stabilizes the unstimulated situation. This is in close agreement with reports about trastuzumab as an anti-cancer therapeutic agent. Trastuzumab is an antagonist of the action of NRG-1 on MCF-7 cells .The above findings as described by visual image analysis are recapitulated in the density/distance plots shown in In As for The CLSM survey images . In this context, it must be stated that the stimulation has an indirect characteristic. The type of agonist and antagonist focus on the manipulation of ErbB-2/3 receptors. Application protocols for agonist and antagonist—like dose and application time—also do not address the requirements for connexin-43. In all reported experiments, the combination of connexin-43 packing density together with the transport into the cytosol are not examined, and this stimulated us to take a closer look at this aspect. In In the CLSM recordings, the mobilization of connexon recapitulates ErbB receptor mobilization at first glance a. A consThe LM localization matrix visual reconstructions in the form of images contain less interpretable information than the connexin-43 protein distance plots given for the plasma membrane d and theThe presented work examines the dynamics of receptor tyrosine kinases of the ErbB family upon stimulation with the canonical agonist neuregulin-1 (NRG-1) and after receptor attenuation by the therapeutic antibody trastuzumab. Additionally, receptor dynamics after DNA-injuring irradiation is recorded. Besides the ErbB-2 receptors as the primary target of examination, the concomitant dynamics of gap junctions after manipulation of ErbB receptors have been recorded. The cell line used for investigations (MCF-7) expresses both ErbB-2 and ErbB-3 receptors, as well as connexin-43-containing gap junctions. After the application of irradiation, NRG-1, or trastuzumab to the otherwise-untouched cells, fixation at life stabilization conditions conserved the cell reaction state. This state was subsequently isolated and identified by indirect immuno-stain and fluorescence microscopy. Consequently, the receptor dynamics described are result of a superposition of life cell stimulation, epitope staining, and CLSM as well as LM microscopy imaging.The modes of increasing the optical resolution during CLSM imaging and circumventing the optical resolution limit by LM matrix recording resulted in particular and individual information about the specimen. Both methods have in common that a conventional image formation by optical interference is replaced by methods with the capacity to acquire fine spatial details of the specimen. CLSM reduces the diffraction of the image recording light spot by cutting off the intermediate image. The accompanying loss of image information is compensated for by scanning the spot across the specimen and recording the image as a line- and frame-wise reconstruction. For LM, an image series is analyzed for spatially- and temporally-isolated fluorescence emission events, and the image is put together from the isolated points as intermittently deposited in a mathematical matrix see .In the two resulting specimen records, the main difference can be assigned as continuous versus discontinuous specimen information. Both representations illustrate certain properties of the ErbB receptor and the connexin-43 constitution and dynamics. While the CLSM displays the continuous aspect of receptor accumulations in plasma membrane and cytosol, it refers to the functional manifestation of ErbB RTKs and gap junctions. Kinase activity and gap junction communication are effective on a In contrast, the results of LM specimen recording identify the set of spatial positions of ErbB receptors and gap junctions as individual manifestations. Each identified protein is displayed as an individual point in a mathematical matrix or in a reconstructed image situation. This approach emphasizes the character of ErbB-2 and connexin-43 as individual protein molecules. The ability to look at the protein distribution in the nm range facilitates the examination of individual inter-protein distances. This information can be correlated with molecular biological stimulation and long-range movement.It can be stated that protein local accumulation, protein long . Cell migration might also be concerned.ErbB receptor plasma membrane accumulation or dissipation and related receptor trafficking in the cytosol after neuregulin-1 or trastuzumab application reflect the cell biological activity of the ErbB-2/3 receptors in transducing the neuregulin-1 stimulation or the suppression of this activity by trastuzumab. Dimerized  ErbB RTKs participate in a set of phosphorylation-based signal cascades. Upon stimulation, cells differentiate, alternate energy metabolism, modulate the levels of stress factors (such as oxidation compounds), and become prone to epigenetic pattern alterations. Activated cascades immediately cover the MAPK, the AKT, and the PKCThe activation or attenuation of ErbB receptors is accompanied by population mobilization, but also by alterations in characteristic population density (or distance) distributions. Receptor density frequently correlates with receptor response amplitude. Increased packing densities of membrane receptors result in a higher signal-to-noise ratio and an accelerated receptor reaction. While this statement meets reality, for example, in the case of electrical membrane channels, the situation after RTK stimulation is more complicated. In the present case, it must be regarded that ErbB-2/3 dimers not only act inside the plasma membrane but are also shuttled to the nucleus to participate in DNA repair regulation. This means that an increase in receptor density might have an ambivalent effect, depending on cell locus and agonist versus antagonist stimulation. Trastuzumab decreases ErbB phosphorylation activity, but increases the plasma membrane fraction density by prohibiting receptor nucleograde trafficking. The activation by NRG-1 and subsequent vesicle-based receptor trafficking induces a depletion of the plasma membrane fraction with an accompanying increase in receptor distances. Further, receptor trafficking across the cytosol results in a broad distance distribution. This reflects the packing of RTKs in transport vesicles.n family (with n = 2 − 4), activation of the kinase function coincides with receptor accumulation. This accumulation covers the basic receptor dimer aggregation with ErbB 2/3, 2/4, and 3/4 heterodimers, and 3/3 and 4/4 homodimers known as carriers for kinase activity [For receptor tyrosine kinases (RTKs) of the Epidermal Growth Factor Receptor (EGFR)/ErbB-activity . In geneactivity . Such agactivity . Additioactivity . In fronactivity . For comactivity .For LM recording, the distance between the emission burst center of the fluorescent dye molecule and the ErbB-2 epitope is a critical prerequisite for close localization. The more distant the dye molecule is, the larger is the uncertainty for the epitope localization. Due to the production process, the secondary antibody as purified from the blood serum of a donor organism directs against a series of epitopes on the primary antibody. Each epitope is located in a non-predictable region on the primary antibody. Further, the dye loading by coupling to serine or glutamate side chain residues anywhere on the secondary antibody further increases the uncertainty for the dye-to-ErbB-2 epitope distance. This situation is sketched in The spatial independence of ErbB-2 epitope location and fluorescence burst location can also introduce both false positive and false negative indicators for receptor clustering. Refer to ErbB receptors are trans-membrane proteins. This means that they can be affected by extracellular signals like NRG-1 or trastuzumab, as well as intracellular signals like tagging, phosphorylation, or complex formation with protein binding proteins like HSP-90, HSP-70, or importin-1The CLSM imaging shows alterations in ErbB protein distribution between the plasma membrane and the cytosol after the application of NRG-1, trastuzumab, or irradiation. In brief, while trastuzumab induces a consolidation of the ErbB membrane fraction, NRG-1 and irradiation stimulate the release of ErbB protein from the plasma membrane into the cytosol. Even more interesting than the mobilization in principal are alterations in receptor density together with receptor trafficking. On the basis of local (membrane vs. cytosol) packing density, a split of the receptor population into different packing classes on the basis of position inside the cell can be seen. The local density dynamics reflect the mode of receptor movement. In the plasma membrane, receptors accumulate in plaques. After plaque formation, the process of membrane bud formation starts the cytosolic receptor retrograde trafficking procedure. The buds form vesicles containing the receptors in characteristic densities and transport them across the cytosol. This procedure can be seen in the pronounced difference when the plasma membrane receptor distance distribution faces the cytosolic one. While at the plasma membrane a broad spectrum of receptor densities for all three cases of manipulation  indicates a minor reaction, the receptor densities in the cytosol are an expression of extended receptor packing activity. Comparing the cytosolic packing density distributions after NRG-1 application with irradiation reveals two individual packing and trafficking modes. While NRG-1 packing covers a broad density distribution, irradiation-induced packing results in a sharp density peak similar to the cytosol receptor residual population in the case of trastuzumab application.This finding gives rise to the assumption that ErbB receptors are subdued to individual packing processes dependent on the mode of receptor activation.The ErbB receptor family members are receptor tyrosine kinases (RTKs). This means that an extracellular stimulus induces an intracellular phosphorylation activity . Thus, EThe appearance of the intracellular membrane apparatus seems to be chaotic with individual vesicles in high density packed in the cytosol, but the process is a highly regulated procedure with protein tags on the vesicle surface for vesicle state and fate . BesidesIn case of ErbB nucleograde trafficking, a terminal point is the fusion of the vesicle with the nuclear envelope and the transfer of receptors across the nuclear pore complexes (NPCs) into the nuclear matrix . The ErbParallels in the mobilization dynamics of ErbB-2/3 receptor dimers and gap junctions shed light on the underlying cytosolic kinase cascades. They also reflect that not only ErbB RTKs, but also gap junctions are key players in the progression and metastatic activity of breast cancer development. In contrast to breast epithelium, gap junctions in the nervous system or in the cardiovascular system have a substantial impact on tissue integrity and function. Such tissues are also subject to ErbB receptor NRG-1-based regulation. The finding of concomitant ErbB and gap junction mobilization by NRG-1-, trastuzumab-, and irradiation-based stimulation poses the question as to whether the systemic release of NRG-1 affects spatial and functional distant cells and tissues in a paracrine fashion. The question of whether accidental irradiation of, for example, heart tissue during anti-cancer therapy induces heart failures must be extended in that the chemotherapeutic manipulation (by trastuzumab) of ErbB receptors in malignant tissue is systematically accompanied by affecting the gap junction distribution in distant non-malignant tissues like the heart. Alterations in cardiac fatty acid turn over and storage, the mitochondria-based energy metabolism, and the signal conducting pathways perturbations are side-effects during anthracyclin/irradiation/therapeutic antibody combination therapy.The dimerization of ErbB-2 with ErbB-3 monomers activates the tyrosine kinase reaction centers on the receptor cytosolic face. One substrate of this ErbB RTK is protein kinase B . This cascade induces the nucleograde transport of RTKs of the Epidermal Growth Factor Receptor (EGFR) type as well as ErbBs . AnotherGap junctions are accumulations of connexons inside regular fields in the plasma membrane . ConnexoDespite their size, gap junctions are highly dynamic aggregates with short half-life and a high lateral mobility inside the plasma membrane ,129,130.Besides the kinase pathway interaction sketched above, the motivation also derives from the role of ErbB-2/4 and 4/4 receptor dimers in the development of the embryonic tissue and from the role of NRG-1 during the differentiation of embryonic stem cells into cardiac myocytes—a procedure accompanied by extensive alterations in the gap junction appearance ,138,139.In several cases, the malignancy of breast cells coincides with a mismatch in the expression and handling of receptors of the ErbB family. This phenomenon is not only synchronized with mismatches in gap junction physiology, but both protein populations turnover and function are closely coupled ,150. ThiMichigan Cancer Foundation cell line 7 (MCF-7) cells were cultivated in Roswell Park Memorial Institute Medium number 1640 (RPMI-1640) with 2 mM L-Glutamine, 100 Units/mL Penicillin, 100 MCF-7 cells were cultivated at 50% confluence prior to NRG-1 application for 12 h in complete RPMI 1640 medium with FCS content reduced to 0.5%. Replace by complete RPMI 1640 medium without FCS for additional 3 h. Replace by complete RPMI 1640 medium without FCS but with 330 nM NRG-1  for 1 h. Replace by complete medium RPMI 1640 with 10% FCS without NRG-1 for one hour before cell fixation.Replace FCS concentration as described above. Replace by complete RPMI 1640 medium without FCS but with 172 nM trastuzumab  for 1 h. Replace by complete medium RPMI 1640 with 10% FCS without NRG-1 for one hour before cell fixation.Cell monolayers at 75% confluence on cover slides in six-well plates (see above for details) were irradiated with 4 Gy total dose by 6 MeV photons using an Artiste linear accelerator . Cells were transported at 37 tion see .w/v) bovine serum albumin fraction V,   and If not otherwise stated, all chemicals were from Thermofisher, Waltham, USA, and phosphate buffered saline (PBS) was used as solvent. For further preparation, the cells were briefly rinsed with PBS supplemented with 1 The Leica TCS SP5 DMI6000 inverted microscope was used with an HCX PL APO lambda blue 63 × 1.4 Oil UV-type objective lens. Four fluorescence acquisition windows (I.–IV.) and one bright field transmission window (V.) were defined as below: I. Excitation 405 nm, acquisition 414–483 nm for nuclear DAPI fluorescence; II. Excitation 488 nm, acquisition 501–551 nm for connexin-43/JGA1 Alexa-488 fluorescence; III. Excitation 561 nm, acquisition 570–623 nm for c-ErbB-2 Alexa-555 fluorescence; IV. Excitation 633 nm, acquisition 642–732 nm for f-actin phalloidin Alexa 647 fluorescence; V. Excitation with accumulated excitation, specimen absorbance acquisition. Frame size 246 In the epi-illumination fluorescence microscope setup, a LightHub-6  boxed laser combining module put the lasers LuxX 405 nm / 60 mW, 488 nm / 200 mW, and 647nm / 140 mW (emission wavelength / light emission power at laser output) onto a common aperture. A beam expanding telescope  fed the beam across a Gaussian to a top hat shaping element  into the rear aperture of a 100 × 1.4 oil immersion objective lens . The epi-illumination path contained three alternative filter sets to discriminate excitation and emission wavelengths with values : 390/40 | 420 | 452/45 and 482/18 | 488 | 525/50 and 642/20 | 659 | 705/72 . The image series  were collected by an emCCD camera  and saved to a PC in 16-bit grey-scale mode in TIFF image stack format. Pixel size was 102 The line plot intensity data from CLSM image recordings were collected using the standard line plot tool in the Image J software package . LM data processing and matrix operations were done in the Matlab software environment in script interpreter mode . In-house designed scripts startSPDM version 4 and Autoclusters were used for matrix generation from image stacks and extraction of distance distribution spectra as histograms. Origin 2015  was used for the generation of envelope function of the distance distribution histograms. The Matlab scripts formulating startSPDM version 4 and Autoclusters are available on request from Michael Hausmann (hausmann(AT)kip.uni-heidelberg.de).


The robotic-assisted laparoscopic retroperitoneal lymph node dissection (R-RPLND) represents a new frontier in the surgical management of testicular cancer in the realm of minimally invasive urologic oncology. We aimed to review the early outcomes as compared to the laparoscopic and open approaches as well as describe the operative technique for the R-RPLND.  We reviewed all the literature related to the R-RPLND based on an electronic PubMed search up until July 2017.  Encouraged by favorable early oncologic and safety outcomes for treatment of clinical stage (CS) I nonseminomatous germ cell tumor (NSGCT), the R-RPLND affords the same recovery advantages as the laparoscopic retroperitoneal lymph node dissection (L-RPLND) while offering greater dexterity, superior visualization, and a theoretically shorter learning curve for the surgeon. While R-RPLND has a promising future in the management of patients with primary and postchemotherapy NSGCT, larger and more vigorous prospective studies are needed before supplanting the open RPLND as the gold standard approach for primary low-stage NSGCT or becoming an equivalent surgical modality in the postchemotherapy setting. Testicular germ cell tumor (GCT) is the most common solid tumor in men between the ages of 20–44. Men diagnosed with GCT have excellent survival rates due to advances in the multimodal treatment paradigm of chemotherapy, radiation therapy, and surgery . RetropeFor primary CS I and CS II NSGCT, minimally invasive RPLND has become a less morbid alternative to the O-RPLND while touting favorable early oncologic outcomes , 6. LapaThe natural evolution of minimally invasive urologic oncology from laparoscopy to robotics laid the foundation for the first robotic-assisted laparoscopic RPLND (R-RPLND) to be performed in 2006 by Davol et al. . The advWe performed an electronic PubMed search for all relevant publications regarding the outcomes and technique of the R-RPLND up until July 2017. We used the keywords robotic, retroperitoneal lymph node dissection, and testicular cancer, which resulted a total of 36 papers. All single and multi-institutional R-RPLND studies in adults with testicular cancer were included and reviewed in addition to studies investigating outcomes associated with O-RPLND and L-RPLND.Patients diagnosed with CS I NSGCT, based on NCCN guidelines, have the option of active surveillance, platinum-based chemotherapy, or RPLND . While eWhile the first use of the L-RPLND was for the purpose of staging alone, the intent of the R-RPLND is to match the oncologic efficacy of O-RPLND while providing the benefits of a minimally invasive approach. The largest R-RPLND series to date, a multi-institutional study of 47 CS I and 5 CS II patients which includes our patient experience, reported an excellent 2-year recurrence-free survival of 97% in the entire cohort . StepaniLymph node yield, a correlate for the extent of node dissection, can provide valuable information regarding the staging and therapeutic benefit of R-RPLND. Importantly, their reported median lymph node yield (LNY) of 26 nodes (IQR I8-32) outperformed the LNY reported in a contemporary meta-analysis of L-RPLND, which reported a median of 16 lymph nodes . R-RPLNDFurthermore, though the oncologic outcomes of the primary RPLND for the management of low-stage NSGCT are often deliberated, there is considerable agreement that RPLNDs should be performed exclusively by experienced high-volume surgeons at experienced institutions, which result in fewer complications and superior oncologic outcomes , 27. TheAs in other minimally invasive surgeries in urologic oncology, the R-RPLND affords a reduced blood loss and shorter recovery time, both of which translate into shorter hospital stays . Blood lPerhaps, the most significant advantage that is afforded by the R-RPLND is the shorter recovery time compared to O-RPLND that translates into a shorter hospital length of stay (LOS). Pearce et al. and Stepanian et al. both reported a median LOS of 1 day which was far superior to both L-RPLND and O-RPLND  , 23. CheMinimally invasive approaches for RPLND, however, are limited by a greater operative time compared to O-RPLND, which persists even beyond the learning curve . In a laAs part of the rationale for primary RPLND to avoid the long-term toxicities of chemotherapy, surgical complications need to be minimized at all costs. O-RPLND, however, has traditionally experienced relatively high intraoperative and postoperative complication rates at 5–7% and 24–33%, respectively . The majPostoperative complication rates are similarly low for R-RPLND compared to open and laparoscopic approaches. Pearce et al. reported only two Clavien Grade 1 complications and two Clavien Grade 3 complications for a postoperative complication rate of 8.5% . While mThe vast majority of low-stage NSGCT patients should obtain a nerve-sparing procedure to preserve antegrade ejaculation for reducing the morbidity of long-term sexual dysfunction. Pearce et al. reported 100% preservation of antegrade ejaculation . SimilarR-RPLND for primary CS I NSGCT involves a transperitoneal approach with the patient typically positioned in the modified flank position with a slightly flexed table. After pneumoperitoneum is achieved with a Veress needle, a 12 mm camera port and three 8 mm robotic ports are placed in a standard linear fashion to triangulate the retroperitoneum. Typically, a 12 mm AirSeal and a 5 mm blunt assistant port are also placed. Others have described a supine or dorsal lithotomy positioning with the patient placed in Trendelenburg and robotic docking occurring over the patient's left shoulder for the daVinci Si or alongside the patient for the daVinci Xi using a four-port linear configuration . A majorPatients are transferred to the floor where their diet is advanced from clear liquids on the night of surgery to a fat-free regular diet on the day after surgery. It is our practice to follow a low-fat diet that is regularly advanced over four weeks to minimize the risk of chyle leak. There is a paucity of data regarding the efficacy of this approach; however, we have not experienced a chyle leak in our institutional experience with minimally invasive RPLND following this protocol. Patients receive education from a nutritionist regarding a low-fat diet (≤20 g fat/day), which they will continue for 4 weeks postoperatively. Patients are usually discharged home on postoperative day 1 after they are tolerating a regular diet and have successfully ambulated and voided with adequate pain control. Patients usually return to school or work within 2 weeks.p=0.03). Notably, two patients required open conversion in the postchemotherapy group, which represents a conversion rate of 22.3%. At a median follow-up of 22 months, there were no retroperitoneal recurrences. Kamel et al., in a series of 12 patients, experienced a 91.7% completion rate with only a single open conversion due to what the author considered poor patient selection [Postchemotherapy RPLND (PC-RPLND) for patients with residual tumors after chemotherapy represents a far more challenging surgery compared to the primary RPLND setting. Desmoplasia from the therapeutic action of chemotherapy can fuse normal tissue planes and add complexity to dissection of tissues and, if needed, repair of vascular injuries. The risk-benefit ratio of cancer cure and morbidity of surgery makes justification of an investigational, minimally invasive technique more challenging. However, select surgeons and centers report perioperative outcomes supporting minimally invasive PC-RPLND as a safe surgery. In addition, perioperative outcomes and complications, including the preservation of antegrade ejaculation, are significantly worse due to the need for a bilateral template and the treatment effect of chemotherapy on the retroperitoneal tissue , 4. Baseelection . At a meelection . TherefoThe first L-RPLND performed in 1992 and the first R-RPLND performed in 2006 marked the beginnings of a minimally invasive era to reduce the treatment morbidity for testicular cancer survivors. Early results from expert robotic surgeons at high-volume academic institutions have demonstrated both feasibility as well as favorable early oncologic outcomes and complication rates in the primary RPLND setting compared to O-RPLND and L-RPLND. Larger, prospective studies are required to better evaluate long-term oncologic outcomes and complication rates in both the primary and postchemotherapy settings.


Agricultural expansion and intensification contributes to global environmental change and risks the long-term sustainability of the planet. It has been proposed that no more than 15% of the global ice-free land surface should be converted to cropland. Bioenergy production for land-based climate mitigation places additional pressure on limited land resources. Here we test normative targets of food supply and bioenergy production within the cropland planetary boundary using a global land-use model. The results suggest supplying the global population with adequate food is possible without cropland expansion exceeding the planetary boundary. Yet this requires an increase in food production, especially in developing countries, as well as a decrease in global crop yield gaps. However, under current assumptions of future food requirements, it was not possible to also produce significant amounts of first generation bioenergy without cropland expansion. These results suggest that meeting food and bioenergy demands within the planetary boundaries would need a shift away from current trends, for example, requiring major change in the demand-side of the food system or advancing biotechnologies.Supplying food for the anticipated global population of over 9 billion in 2050 under changing climate conditions is one of the major challenges of the 21 Projected population growth to over 9 billion people by 2050, the impacts of climate change on agricultural productivity –4, and sth century, while for the most part agricultural intensification was responsible for increased production, further expansion of agricultural areas still occurred [Historically, cropland expansion into forest and other unmanaged natural areas has met increased demand for land-based commodities . Since toccurred ,12. Cropoccurred  suggesteoccurred . Crop cuoccurred –17. Climoccurred ,19. Curroccurred –22. Howeoccurred .Taken together there are large pressures on the land system to feed a growing population, to contribute to climate mitigation, and to nonetheless stay within the planetary boundary for cropland area. Over recent decades, scenario analyses have played a central role in assessments of addressing such growing pressures, seeking to explore the potential effects of socioeconomic change and global environmental change. Such scenarios are derived from an interpretation of coherent, qualitative storylines describing future pathways or projections –27. ExplIn a normative  scenario approach, targets representing a desired situation at some point in the future are first defined and pathways to achieve these targets are interpreted or derived, often from model simulations . Existin−1 of the global primary energy supply in 2050 was supplied by bioenergy. For the bioenergy-mitigation target we therefore take a conservative approach and set the contribution of first generation bioenergy crops to 9 EJ by 2050. This aligns with the bioenergy pathway projected in the world energy outlook [2eq, resulting in a 50% likelihood of keeping the global average temperature increase below 2°C compared to pre-industrial levels.In this paper, we explore whether meeting these three multiple objectives is feasible in the future. Unlike similar modelling frameworks the Parsimonious Land-Use Model used here has rapid runtimes and thus allows for multiple model iterations to explore across parameter space within plausible uncertainty ranges . Given t outlook  450 scen-1 d-1; [-1 d-1 on average per country.The food-supply target should ensure adequate food supply for the entire global population. Dietary requirements were estimated from a global population of 9.1 billion in 2050  and a da-1 d-1; ). The pe-1 d-1;  used her-1 d-1;  and so, The planetary boundary target for global cropland area assumes that up to 15% of the global, ice-free terrestrial surface can be used for crop production ; the curFor the bioenergy-mitigation target we chose the bioenergy pathway projected in the 450 scenario from the world energy outlook . Given uThe Parsimonious Land-Use Model was used to simulate the agricultural system until 2050 . Parameters held constant and justification for doing so can be found in the Population, income levels and diet drive the demand for agricultural products as previously described in Engström et al. . CalculaUsing the parameter values from 2 fertilization effect. To ensure consistency between the energy scenarios underlying the bioenergy projections and the climate scenarios underlying the crop yield projections, different yield projections were used for the first and second set of runs. For the first set of runs we assumed higher levels of climate change and so, simulated crop yields based on climate data for RCP6.0 [2eq, we simulated crop yields based on the more stringent climate mitigation scenario, RCP2.6 [Crop yield inputs to PLUM were simulated with the global dynamic vegetation model LPJ-GUESS ,53  that fulfilled: a) the food-supply target, b) both the food-supply and cropland planetary boundary targets and c) all three targets: the food-supply, cropland planetary boundary, and bioenergy-mitigation target. From these individual model parameterisations, we created output narratives to describe the key elements of the model runs that were instrumental in achieving the normative targets.-1 over the 2000–2050 period.The food-supply target alone can be met by a large number of different simulations , but mosAlthough no model runs achieved all normative targets, three model parameterisations came close to this. Of these, two model runs, named FC-lowNV and FC-highNV, achieve both the food-supply and cropland planetary boundary targets, but not the bioenergy-mitigation target. The third model run, named FB-highNV, fulfils the bioenergy target however, global cropland expansion surpasses the cropland planetary boundary target by 2050. Parameter settings for the three scenarios can be found in FC-lowNV only 7% of potential arable land is conserved as natural vegetation at a country level . However, this is only achieved by global cropland expansion surpassing the cropland planetary boundary target by 2050. The global cropland area is 2082 Mha in 2050, which is 15.5% of the total ice-free land area (-1 d-1 in 2000) in FC-lowNV. For FC-lowNV the increase in cereal consumption for countries with low initial energy supply is 13% by 2050 compared to 2000, while it is 6% for FC-highNV. In PLUM, milk and meat consumption are proxies for rising incomes shifts to protein-rich, high-fat diets. This relationship is well established in the literature [In general, meeting the food-supply target required a strong increase in meat and milk consumption in emerging and developing countries  and thus a continuation/expansion of Western diets. Only small differences with respect to consumption patterns exist between FC-lowNV and FC-highNV. In 2050, simulated average meat consumption in developing countries in FC-lowNV is 10 kg meat per capita lower than the average meat consumption in FC-highNV. The lower consumption of animal products in FC-lowNV is balanced by an increase in cereal consumption for countries with very low initial food supply . The difference in global average yield increase, combined with different values for the conservation of natural vegetation lead to different patterns in cropland change at the country scale  not accounting for double cropping by deriving global average yield  and (2) including cropland degradation assumptions that result in production losses, decreasing global average yields.PLUM derives crop yields from the LPJ-GUESS by making assumptions about the role of technological development and improved land management ,47. Ther-1 over the 2000–2050 period. Other studies have concluded that yield increases of more than 1% per year are needed [-1 over the 2000–2010 period, and historical yield growth of, for instance, 2.5% yr-1 over 1961–1991 , these  are similar or even higher (58% and 68% in 2000–2050 for FC-lowNV and FC-highNV respectively) compared to the values used by Haberl et al. [Comparing bioenergy potential across studies can be misleading because of differences in bioenergy terminology , but also because of different assumptions regarding feedstock  and the availability of technology . Consequently, within the existing literature there are vast differences in estimates of bioenergy potential ,40,71–75l et al.  in the Bl et al.  reportedl et al. . Accountl et al. .Projections for both bioenergy demand and food demand vary greatly. For example, Dornburg et al.  showed eIn general, meeting the food-supply target required a strong increase in meat and milk consumption in emerging and developing countries and thus a continuation/expansion of Western diets. None of our simulations with lower meat consumption met the 2050 food-supply target. However such projected protein-rich, high-fat diets pose potential health threats to countries, arising from obesity and associated poor health . FurtherMeeting the food supply and bioenergy targets required cropland expansion beyond the cropland planetary boundary and the expansion of cropland into areas of natural vegetation in the tropics found in all scenarios would cause changes in local climate and contribute greatly to emissions from the land use sector ,79. We dAn alternative to enhance sustainability in the food sector is the reduction of food waste ,86. At tThe production of adequate food supply within the cropland planetary boundary under current population trends, economics and diets is only possible with annual crop yield increases of at least 1%. Achieving yield increases requires a reduction in the yield gap in all food producing countries with potential implications for the sustainability of agricultural ecosystems. Achieving bioenergy production from first generation energy crops of an order that would contribute to strong climate mitigation is however not possible at the same time. Yet, recent work indicates that meeting multiple food and energy security goals in a sustainable way could be achieved by a reduction in food waste or the widespread uptake of sustainable diets.S1 File(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S1 FigParameter settings for simulations that achieve the food supply target (blue), and simulations that additionally are within the cropland planetary boundary target (yellow), as well as the parameterisation that also achieves the bioenergy target, but is only almost within the cropland planetary boundary (red).(TIF)Click here for additional data file.


Our analysis further shows that daf-7 and tph-1 contribute to most of the food-responsiveness in the modulation of lifespan. We applied a computational model to capture the general coding features of this system. This model agrees with our previous genetic analysis and highlights the consequences of redundancy and synergy during information transmission, suggesting a rationale for the regulation of these information processing features.Neuroendocrine circuits encode environmental information via changes in gene expression and other biochemical activities to regulate physiological responses. Previously, we showed that se genes . Here, wDOI:http://dx.doi.org/10.7554/eLife.24040.001 The lifespan response to food abundance consists of tph-1(-); daf-7(-) double mutant  animals retain redundancy by compensating this effect with an increase of signal correlation; however, this balance shifts in the daf-7(-) mutant due to the dramatic reduction of signal correlation (tph-1(-); daf-7(-) double mutant has nearly equal signal and noise correlations which generate independent encoding.Multicellular coding strategies rely on response correlations between cells . Specifirelation , bringinrelation . The tphdaf-7 and tph-1 over a biologically relevant range (daf-7 in noise reduction (tph-1(-) mutants displayed increased sensitivity to both noise and correlations. Redundancy in daf-7(-) mutants was more sensitive to correlation than noise, a reversal of the wild-type situation. tph-1(-); daf-7(-) double mutants were less sensitive to noise and correlations than either single mutant. These results suggest that the sensitivity of redundancy to noise is controlled by daf-7, while robustness to correlation is maintained by both daf-7 and tph-1.Redundancy and synergy is strongly affected by noise and correlation among neurons. To characterize their effects, we rescaled noise and correlations in the original response distributions of eduction . tph-1 mutants show reduced signal-to-noise, and adopt synergistic encoding (tph-1(-); daf-7(-) double mutants compared to daf-7(-) single mutants  mutants, because wild-type animals employ redundancy, whereas daf-7(-) mutants employ a synergistic encoding. Indeed, we find that the ability to accurately discriminate between different food inputs based on lifespan is degraded in daf-7(-) mutants  and tph-1(-) animals, as the mutations do not affect the development of the ASI, ADF, and NSM neurons, which remain food-responsive. Instead, we show that daf-7 and tph-1 influence information processing via effects on the signal and noise properties of these sensory neurons, and on their correlations, representing additional roles for these genes in controlling information encoding. The discovery of other genes that regulate the signal-to-noise ratio will likely provide further insights into genetic regulatory mechanisms that modulate neural coding.By extending the analysis of our previous work, we have found that the ADF, NSM and ASI neurons employ a redundant strategy to encode food information. Critically, this redundant encoding strategy is controlled by C. elegans transgenics. Using fluorescent reporters also eliminates experimental noise associated with antibody staining due to variability in fixation, in permeabilizing the C. elegans cuticle, and in signal amplification from secondary antibodies. Third, we minimized bleaching by using a combination of low excitation from an LED light source, and rapid image acquisition using a Piezo Z stage (Prior Scientific) that precisely moves the sample in the Z axis at high speed.Information theory relies on accurate estimates of response distributions, requiring the minimisation of experimental variability. We took several steps to achieve this. First, we only considered animals oriented in a dorso-ventral position. The microfluidic chip was constructed to bias animals towards this correct orientation, the orientation was checked during automated cell identification and verified manually, ensuring that only image stacks with animals in dorso-ventral orientations were used in the analysis. Second, we used direct imaging of transcriptional fusions to fluorescent protein reporters integrated in single copy. This approach ensures that biological variance in promoter activity is not artificially washed out by averaging in conventional high-copy reporters that are more traditionally used to generate Pdaf-7::mCherry and Pdaf-7::Venus reporters integrated at single copy in precise genomic locations on LG I and LG II, respectively . Mathematical details of these procedures and the results are discussed in the Appendix.The computational analysis of all the data was performed using custom-made C++ programs and built-in implementations of standard multivariate analysis algorithms in R . C++ pro In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor (Oliver Hobert) and a Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Genetic Control of Encoding Strategy in a Food-sensing Neural Circuit" for consideration as a Research Advance by eLife paper.The reviewers have discussed the reviews with one another and are in agreement that this work qualifies as an interesting Research Advance to your original The only comment we would like you address is extremely minor in nature:


The Contrast Media Safety Committee (CMSC) of the European Society of Urogenital Radiology (ESUR) has updated its 2011 guidelines on the prevention of post-contrast acute kidney injury (PC-AKI). The results of the literature review and the recommendations based on it, which were used to prepare the new guidelines, are presented in two papers.Topics reviewed include the terminology used, the best way to measure eGFR, the definition of PC-AKI, and the risk factors for PC-AKI, including whether the risk with intravenous and intra-arterial contrast medium differs.PC-AKI is the preferred term for renal function deterioration after contrast medium.• PC-AKI has many possible causes.• The risk of AKI caused by intravascular contrast medium has been overstated.• Important patient risk factors for PC-AKI are CKD and dehydration.•  The Contrast Media Safety Committee (CMSC) of the European Society of Urogenital Radiology (ESUR) produced their most recent guidelines on what was then termed contrast induced nephropathy (CIN) in 2011 . This reThe clinical features and incidence of PC-AKI.The choice of terms for renal function deterioration after CM, the degree of renal function deterioration used to diagnose PC-AKI, and the definitions of intravenous and intra-arterial CM administration.The reliability of the various equations used to measure eGFR and the appropriate timing of eGFR measurement before CM administration.The evidence that CM can cause AKI, the levels of renal function at which there is a risk of PC-AKI, and the recent evidence suggesting that the risk of PC-AKI may be lower after intravenous than after intra-arterial contrast medium.The importance of the many risk factors for PC-AKI described in the literature.In this first paper on PC-AKI, the following topics related to diagnosis and risk are considered:Recommendations are made for items 2–5. The recommendations have been incorporated into version 10 of the ESUR CMSC guidelines  CM has probably been overestimated. Two meta-analyses of 19,000 patients who had received IV CM showed PC-AKI incidences of 6.4 % (95 % CI 5.0–8.1) and 5.0 % (95 % CI 3.8–6.5) , 12. In It has been suggested that intra-arterial (IA) CM administration during catheter-based angiography, with or without percutaneous coronary intervention (PCI), is associated with a higher incidence of PC-AKI than IV CM administration , 14. HowThe guidelines were developed using the Appraisal of Guidelines for Research and Evaluation (AGREE) II document . A guideThe titles and abstracts were screened for relevance and selected on predefined in- and exclusion criteria. Emphasis was put on comparative studies with strong scientific evidence, such as meta-analyses and systematic reviews, and prospective randomized controlled trials (RCTs). In addition, evidence was collected from comparative cohort, patient-control and non-comparative studies. Other important quality criteria were size of population studied, duration of follow-up and control for bias. Cross-referencing was used to find additional data. The four systematic searches for paper 1 yielded 3,086 references, of which 705 were selected from their title and abstract. The full texts of these 705 publications were reviewed and 105 were selected for inclusion in this paper. The quality of evidence was evaluated according to the Oxford Centre for Evidence Based Medicine (OCEBM) 2011 levels of evidence: Grade A: established scientific evidence; Grade B: scientific presumption; Grade C: low level of evidence . Where tOther factors such as availability of techniques or expertise, organizational consequences, financial costs and patient preferences were also considered. CM manuals and guidelines (American College of Radiology (ACR), Royal Australian and New Zealand College of Radiology (RANZCR), National Institute for Health and Care Excellence (NICE), Royal College of Radiologists (RCR), and Radiological Society of the Netherlands (RSTN)) were consulted where appropriate..The recommendations prepared by the WG are the result of the available scientific evidence combined with these other sources of information. They were discussed at the CMSC meeting in Copenhagen, Denmark in February 2017 and the text of the final recommendations and guidelines was subsequently approved by the academic members of the CMSC. Once published in print, the validity of the CMSC guidelines will be routinely set at 6 years. However, the CMSC members constantly monitor the validity of the guidelines, and can propose revision at an earlier date if deemed necessaryUntil recently there has not been a generally accepted term for acute renal failure, which is a complex disorder with many possible causes and risk factors. Several nephrology groups, such as the Acute Dialysis Quality Initiative (ADQI)  and Kidnpost-contrast acute kidney injury (PC-AKI) is a general term that should be used if there is a sudden deterioration in renal function within 48 h of the intravascular administration of iodine-based CM. They describe PC-AKI as a correlative diagnosis. They recommend that the term contrast-induced acute kidney injury (CI-AKI) is reserved for cases where a causal relation can be shown between the administered CM and the deterioration in renal function. However, in clinical practice it is usually difficult to distinguish CI-AKI from PC-AKI and very few of the published studies have a suitable control group to allow the two conditions to be separated. Thus, many cases of PC-AKI seen in clinical practice or reported in clinical studies are likely to be coincident to, but not caused by, CM administration.The CMSC recommends that the term PC-AKI should replace the older term of contrast-induced nephropathy (CIN) and suggests using the terms recommended by the ACR Committee on Drugs and Contrast Media  when AKIThe diagnosis of PC-AKI is usually based on surrogate measures of absolute or relative change in serum creatinine (sCr), rather than patient outcomes, such as renal failure, need for RRT or mortality. The KDIGO Practice Guidelines , 27 adopin the absence of an alternative aetiology” [The ESUR CMSC defined Contrast-Induced Nephropathy (CIN) in their first survey-based guideline as “a condition in which an impairment in renal function  occurs within 3 days following the intravascular administration of a contrast medium tiology” .2) and CKD 2 (eGFR 60–89 ml/min//1.73m2), and absolute values seem to be more sensitive for patients with CKD 3A (eGFR 45–59 ml/min/1.73m2) [Multiple studies have shown that the incidence of PC-AKI is largely dependent on the definition used –31. A re/1.73m2) , 34. Stu/1.73m2) .The KDIGO criteria are more rigorously derived than the CIN definition and are now being adopted as the standard for PC-AKI studies . The CMSintravenous CM administration indicates that CM reaches the renal arteries after dilution by circulation through the right heart and pulmonary circulation or a systemic capillary bed.The term intra-arterial CM administration with second-pass renal exposure, such as via catheters into the right heart and pulmonary arteries and via catheters directly in the carotid, subclavian, brachial, coronary and mesenteric arteries, as well as into the infrarenal aorta and the iliac, femoral and crural arteries. Note: Because of backflow during this type of IA injection, small doses of CM may reach the kidney in a relatively undiluted form.The same is true for ntra-arterial CM administration with first-pass renal exposure indicates that CM reaches the renal arteries during its first pass in a relatively undiluted form, depending on the distance of the site of injection from the renal arteries. This occurs with injections through catheters into the left heart, the thoracic and suprarenal abdominal aorta, and selectively into the renal arteries. Note: In suprarenal aortic injections, some of the injected CM escapes via suprarenal aortic side-branches and reaches the kidney after dilution in the circulation.The term iTotal glomerular filtration rate (GFR) is considered the best overall index of kidney function, but cannot be measured easily in clinical practice, so GFR is estimated using sCr as an endogenous filtration marker. In 1999, the Modification of Diet in Renal Disease (MDRD) equation  was intrIn 2009, the CKD-EPI equation was proposed by the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI), and was shown to be superior to the MDRD equation, especially at higher GFRs Table . All creThere are other equations for specific subgroups, such as the Lund-Malmö (LM) revised equation for the Swedish population , the BerWhen estimating GFR in children, sCr levels should be measured by standardized reference methods because serum concentrations are lower than in adults . The CMSA Cystatin C-based equation has been proposed that showed the best accuracy (91 %) when combined with height/SCr, height, sex and blood urea nitrogen (BUN) . HoweverPoint-of-care (PoC) whole blood creatinine may be measured with the older Jaffe  method or by enzymatic methods, with the latter considered more accurate. Although such measurements have practical advantages in patients with increased risk of PC-AKI, PoC devices may lead to overestimation of renal function in severe kidney failure with resultant incorrect risk stratification . LaboratThere are no studies available on how long eGFR measurements remain valid for estimating the PC-AKI risk. The eGFR measurements can be regarded as stable in individuals without CKD or underlying co-morbidities such as heart failure or hypertension who are not taking nephrotoxic drugs.a maximum of:has an acute disease, an acute deterioration of a known chronic disease or any other adverse event that could have negatively influenced renal function (eGFR), oris a hospital inpatient7 days* if the patientif the patient has a chronic disease with stable renal function (eGFR), andin all other patients Table 3 monthsThe CMSC considers eGFR measurements before intravascular CM exposure valid for *Note: In patients with AKI, eGFR should be monitored frequently, and a maximum of 1–2 days is advisable.Contrast-induced nephropathy was accepted for many years, but more recently it has been questioned whether CM causes the deterioration in renal function that may occur after CM administration , 53. TheThere is controversy about the causal relationship between exposure to IV CM and PC-AKI, since there are no prospective RCTs confirming this association , 57. WitBased on comparisons of the relatively few studies with and without control populations it has been suggested that the risk of PC-AKI after IV CM has been overestimated , 59. A m2, but there is conflicting evidence on whether patients with severe renal impairment (eGFR <30 ml/min/1.73m2) are at increased risk of PC-AKI [Evidence from observational studies may need to be used, despite the recognised methodological problems . Recentlf PC-AKI , 65. Lacf PC-AKI . The faiA limited number of studies have directly compared IV to IA CM administration, using the patient as their own control. The risk of PC-AKI as well as its clinical course was independent of the route of administration in four retrospective studies of patient populations with varying degrees of renal impairment –70, and The PC-AKI incidence following direct IA CM administration with first-pass renal exposure is frequently reported to be higher than after IV administration, but this remains controversial , 72. ProSince it is difficult to separate the effects of the procedure from those of the CM, the CMSC decided, for optimal safety, to choose a higher cut-off eGFR level for preventive measures in patients undergoing catheter-based diagnostic or interventional examinations using IA CM administration with first-pass renal exposure, even though some of the risk may relate to the procedure. Also, the CMSC decided to include CA and/or PCI in this category because these examinations frequently combine IA CM administration with both first- and second-pass renal exposure Table .Table 4R2 [There is limited evidence about PC-AKI in several special populations, such as patients with renal or renal and pancreatic transplants, or critically ill patients. In renal transplant recipients, the incidence of PC-AKI in patients receiving either IV or IA CM was not higher than in patients without transplants, and there was no graft loss or need for dialysis –77. Crit2 .There are very few studies on paediatric PC-AKI –85. As tImpaired renal function is the most important patient risk factor for PC-AKI. Many meta-analyses and systematic reviews of uncontrolled studies have identified a wealth of possible clinical risk factors for AKI in general such as old age, female gender, low BMI, classic cardiovascular and metabolic risk factors, malignancy, inflammation, bleeding, anaemia and hyperuricaemia , 86–96. A variety of risk factors have been related to the type of CM and the way it is administered.Multiple meta-analyses have shown no evidence that iso-osmolar CM (IOCM) are associated with a significantly lower rate of PC-AKI than non-ionic, low osmolar CM agents (LOCM) –100. Howabsolute eGFR  below 1.1 [2) below 3.0 when using a CM concentration of 350 mgl/ml [Evidence about the influence of CM dose (CM volume x CM concentration) indicates dependence on the route of administration. There is insufficient evidence that dose is a problem with IV CM. However, for direct IA CM administration in coronary angiographic intervention it is advisable to keep the ratio of CM dose (in grams Iodine) to elow 1.1 , 103 or 0 mgl/ml , 105 (TaPC-AKI has been adopted as the best term to apply to renal function deterioration after intravascular CM administration because, unlike some of the older terms, it does not imply that CM is the cause. Stage 1 of the KDIGO classification of AKI is recommended as the change in renal function used to diagnose PC-AKI. The principal risk factor for PC-AKI is impaired renal function, and the recommended ways to measure this are by the CKD-EPI equation in adults and the Schwartz equation in children. In recent years, it has become apparent that the risk of true CI-AKI was overstated in the past. When properly corrected for the many other possible causes of AKI in patients with chronic kidney disease, the risk of CI-AKI when modern low osmolar CM are administered IV or IA is low. Repeated CM administration within a 24- to 48-h period increases the risk of CI-AKI. The evidence of a higher risk with IA than with IV CM administration is limited, but the CMSC nonetheless considers that the cut-off levels of eGFR used to indicate the need for prophylaxis before IA administration with first-pass renal exposure should be stricter, and that there should be a maximum volume of CM given intra-arterially during any examination or procedure with first-pass renal exposure.The recommendations made in this paper have been incorporated into the ESUR CMSC guidelines (see Table