The data, consistent with other independently developed tree-ring-based hydrological records from cold semi-arid to arid NW Himalaya and Karakoram, point to an increased regional wetting in the recent decades. Such an increased wetting might have led to the anomalous behaviour of glaciers in the NW Himalaya and Karakoram in contrast to the general receding trends in the central and eastern Himalaya.Hydroclimatic variability driven by global warming in the climatically vulnerable cold semi-arid to arid northwest (NW) Himalaya is poorly constrained due to paucity of continuous weather records and annually resolved proxies. Applying a network of annually resolved tree-ring-width chronologies from semi-arid region of Kishtwar, Jammu and Kashmir, India, we reconstructed April-May standardized precipitation index extending back to A.D. 1439 (576 years). The reconstructed series is featured by the most conspicuous long-term droughts during the 15 Most of the glaciers in different parts of the Earth show receding trends owing to global warming, however, some glaciers in the NW Himalaya and Karakoram show anomalous trend with stability or mass gain in the recent decades8, accentuating the importance of changing regional precipitation patterns9 in response to global climate change. Nevertheless, our understanding on spatio-temporal variability of precipitation, as one of the most important drivers for the cryospheric change, remains poorly constrained due to non-availability of long and continuous precipitation records10. We investigated April-May standardized precipitation index variability in semi-arid Kishtwar in the NW Himalaya reconstructed from a network of annually resolved Neoza pine  and Himalayan cedar (Cedrus deodara (Roxb.) G. Don) ring-width chronologies from a network of moisture stressed sites. The precipitation records of Kishtwar (1901–50) show that ~66% of the annual precipitation occurs from December to May of which ~27% falls in April-May alone, connoting its significant contribution to the regional hydrology.Glaciers in the NW Himalaya and Karakoram, a perennial source of stream recharge, largely sustain on winter and spring (December–May) precipitation brought by mid-latitude westerlies17. Tree-ring-based precipitation records from cold semi-arid to arid NW Himalaya and Karakoram have revealed increased pluvial conditions in the 20th century20. However, hitherto Jammu and Kashmir in the NW Himalaya represents a data void, and thus annually resolved tree-ring records are essentially required to understand hydroclimatic variability across the north-south transect from Karakoram to the NW Himalaya where major part of annual precipitation occurs in winter and spring under the influence of mid-latitude westerlies. Tree-ring-based hydroclimatic proxy records25 from Jammu and Kashmir are short and do not show significant variations on inter-decadal time scale. Such studies in this region after the initial attempts in the 1980s29 could not take pace further largely due to lack of logistics and difficulties experienced in approaching remote locations, where pristine old forest stands are found. Here, we present analyses of standardized precipitation index of April-May (SPI2-May) reconstructed for the first time using network of annually resolved tree-ring data of Neoza pine and Himalayan cedar from semi-arid Padder Valley of Kishtwar, Jammu and Kashmir, NW Himalaya. The data were compared with other proxy records available from cold semi-arid to arid regions of the NW Himalaya and Karakoram to understand spatio-temporal variability in droughts vis-à-vis the state of glaciers in long-term perspective. High-resolution archives of hydroclimate from a close network of orographically varied Himalayan regions, as presented here, should also be useful in assessment of sensitivity of precipitation to a range of forcing factors on different timescales and also evaluate climate model skills in projecting future trend of climate variability30.Observational precipitation records from the NW Himalaya and Karakoram, though very short and scanty, are restricted to valley floors, and show mixed, both increasing/decreasing trends that indicate orography-forced high spatial variability32. The Neoza pine trees usually prefer to colonize very dry rocky settings and for such ecological preferences it often grows pure and scattered forming open canopy forest. The competition among trees in such open stands is low making them a sensitive sensor of hydroclimatic variability. The growth pattern analyses of trees from open stands indicate radial growth increase in recent decades, especially since 1970s  distributed over an altitude range of 1900–2400 m asl in Kishtwar  is low, contributing ~16% of the annual precipitation 41 and Standardized Precipitation Index (SPI)42. Pearson correlation analyses of SPI with tree-ring chronologies (figure not shown) as well as PC#1 showed highest correlation with the SPI of April and May (SPI2-May) . Calibration and verification statistics, such as Pearson correlation coefficient, sign test, reduction of error (RE), and coefficient of efficiency (CE)44 denoted statistical skill in the reconstruction (Supplementary Table 40 available through the KNMI climate explorer (https://climexp.knmi.nl45), and PDSI of Monsoon Asia Drought Atlas (MADA46). The SPI2-May reconstruction showed consistency with the corresponding month’s drought variability over large parts of the Himalaya-Karakoram and Central Asia  of ring-width chronologies  for the common period A.D. 1717–2014 indicated high common variability and strength of common forcing factor  from A.D. 1439–1660s and positive excursions (wetter climate) since A.D. 1670s onwards. The long-term pluvial phase from 1670s to 2014 was also punctuated by relatively drier periods of 1850s–1870s and 1970s. A comparison of SPI2-May reconstructed series with precipitation records developed from cold-arid regions of Lahaul19 and Kinnaur20 in the NW Himalaya, where annual precipitation variability is largely controlled by the mid-latitude westerlies in winter and spring, revealed strong consistency in variations over inter-annual, inter-decadal and centennial timescale . Distinct consistency in tree-ring-based hydroclimatic records from the NW Himalaya and Karakoram  50 could also be linked with the increasing trend in precipitation in the region. The stability/advancement of many large glaciers in Lahaul51 and Karakoram8 could be associated with the increased import of moisture by the mid-latitude westerlies in recent decades. The reduction in run-off of Hunza and Shyok river flow during 1961–2000 has also been implicated to glacier stability, which has been found to be largely due to increasing winter precipitation13. These features are also consistent with the descending trend in modelled equilibrium-line altitude reported in Karakoram during 1976–199552. The glacier thickening in the northwest Himalaya and Karakoram is in agreement with the increasing trend in precipitation11, but strikingly in contrast to mass balance studies from the central and eastern Himalaya56 where glaciers are in recession phase.The most conspicuous feature of the reconstructed drought indices is an increase in SPI values in the late 20th century featured by pluvial conditions was also punctuated by drier 1970s with 1970 and 1971 being very dry. The hydrological records available from semi-arid regions of the NW Himalaya show that these droughts were wide spread in cold-arid regions of Lahaul-Spiti57 and Kinnaur20. The extended large-scale droughts of 1970s could have caused serious stress on socio economy of the Kishtwar region. The droughts of 1970–71 were widespread in Afghanistan, Pakistan and Tajikistan as well, largely due to failure of winter snowfall triggering population displacement, loss of cattle and severe food shortages58. To understand the causal factors of such droughts we investigated relationship of SPI data with North Atlantic Oscillation (NAO) and El Nino Southern Oscillation (ENSO), which are prominent modes of variability exerting strong influence on precipitation in southwest central Asia and north India60. The positive phase of NAO and warm phase of ENSO are associated with positive anomalies in precipitation, however, the former has strong linkage with precipitation variability in winter only when it is most active60. Our SPI2-May data did not reveal significant relationship with NAO except weak correlation with the indices of April only . We observed that the SPI data are directly associated with temperature anomalies in the tropical eastern Pacific Ocean and Indian Ocean  statistics commonly practiced in dendroclimatology37.The tree-ring samples of Neoza pine and Himalayan cedar in the form of increment cores were collected from moisture stressed sites in Padder valley, Kishtwar, Jammu and Kashmir, NW Himalaya. Each growth ring in the samples was assigned to the calendar year of its formation by using established procedures of crossdating. The ring-widths in precisely dated increment core samples were measured to the nearest 0.01 mm using a LINTAB system . The ring-width measurement series were standardized using appropriate detrending methods to maximize the common variance and remove non-climatic growth trends. In this process, signal free detrending42.As weather records from any station close to tree ring sites is not available, we prepared regional precipitation series by merging monthly precipitation data of eight weather stations in Jammu and Kashmir  back in time were used in the reconstruction. The PC#1 with an eigenvalue >1 of each nest showing a significant positive relationship with SPI2-May  was used in calibration and reconstruction. The split period calibration in 1907–1926 and 1927–1946 approach was performed to test the fidelity of relationship between PC#1 and SPI2-May. Due to short length of observed SPI2-May series of only 40 years (1907–1946), we also performed leave-one-out cross-validation66 to test the validity of calibration model. After establishing the fidelity of calibration model we used 1907–1946 regression model to develop the final reconstruction for each nest. Accordingly, four reconstructions of different lengths involving a varying number of predictor chronologies in different nests were developed. The nested reconstructions developed using the full period (1907–1946) calibrations were spliced together to develop the final reconstruction extending from A.D. 1439 to 2014. To minimize possible artifacts associated with the changes in variance through time due to decreasing number of predictors, the mean and standard deviation of each nested series was scaled to that of the most replicated nest one (A.D. 1717–2014) prior to averaging.The network of five ring-width chronologies  was used to calibrate and reconstruct SPI2-May. At first, the predictor chronology variables (t0 and t + 1) were tested for their relationships with SPI2-May. We observed that only t0 variable of the chronologies passed the threshold correlation significance level (p ≤ 0.01). As the number of available tree-ring chronologies decreased back in time we used a nested approachSupplementary Information


We report the generation of a preclinical IBC patient-derived xenograft (PDX)-derived ex vivo (PDXEx) model and show that it closely replicates the tissue architecture of the original PDX tumor harvested from mice. The gene expression profile of our IBC PDXEx model had a high degree of correlation to that of the original tumor. This suggests that the process of generating the PDXEx model did not significantly alter the molecular signature of the original tumor. We demonstrate a high degree of similarity in drug response profile between a PDX mouse model and our PDXEx model generated from the same original PDX tumor tissue and treated with the same panel of drugs, indicating that our PDXEx model had high predictive value in identifying effective tumor-specific therapies. Finally, we used our PDXEx model as a platform for a robotic-based high-throughput drug screen of a 386-drug anti-cancer compound library. The top candidates identified from this drug screen all demonstrated greater therapeutic efficacy than the standard-of-care drugs used in the clinic to treat triple-negative IBC, doxorubicin and paclitaxel. Our PDXEx model is simple, and we are confident that it can be incorporated into a PDX mouse system for use as a first-pass screening platform. This will permit the identification of effective tumor-specific therapies with high predictive value in a resource-, time-, and cost-efficient manner.Inflammatory breast cancer (IBC) is a rare and aggressive presentation of invasive breast cancer with a 62% to 68% 5-year survival rate. It is the most lethal form of breast cancer, and early recognition and treatment is important for patient survival. Like non-inflammatory breast cancer, IBC comprises multiple subtypes, with the triple-negative subtype being overrepresented. Although the current multimodality treatment regime of anthracycline- and taxane-based neoadjuvant therapy, surgery, and radiotherapy has improved the outcome of patients with triple-negative IBC, overall survival continues to be worse than in patients with non-inflammatory locally advanced breast cancer. Translation of new therapies into the clinics to successfully treat IBC has been poor, in part because of the lack of  Inflammatory breast cancer (IBC) is a rare, clinically and pathologically unique breast cancer subtype  that accVan Laere et al  showed tin vitro cell culture models that fail to recapitulate the complex 3-dimensional (3D) tumor microenvironment as screening platforms to identify new drugs [Ex vivo culture models [One of the reasons for the lack of success to date in identifying effective therapies for triple-negative IBC is the use of simplified ew drugs –37. Anotew drugs . Althougew drugs –45, thesew drugs . Currentew drugs . PDX modew drugs , 47. PDXew drugs , 48. Ex e models  and orgae models –50 are cex vivo (PDXEx) model generated from the cellular milieu released from a PDX tumor harvested from a mouse. Our PDXEx model closely replicates the tissue architecture of the original PDX tumor. We show a highly significant correlation between the gene expression signatures of the ex vivo tumor tissue model and the original PDX tumor, suggesting that our PDXEx model retains certain complex intra- and extra-cellular communication networks, cell signaling pathways, and differentiated cell types characteristic of the in vivo tumor and thus will have physiological responses similar to those observed in the in vivo model. We confirm the value of our model in terms of predicting effective tumor-specific therapies and demonstrate its potential application as a drug screening platform to identify novel and effective IBC tumor-specific therapies. Generating the PDXEx model was time, resource, and cost effective, and we predict that incorporating this PDXEx model as a first-pass screening platform within a PDX system will permit the cost- and time-efficient identification of therapies with a high probability of efficacy against the original tumor. Combining the PDXEx model with a PDX system has the potential to identify new therapies that would result in benefit if incorporated into an individual patient’s adjuvant therapy regimen.Herein, we chronicle the development of an IBC PDX-derived Advanced DMEM (Gibco Life Technologies) was supplemented with Pen/Strep (50 units/ml), antibiotic/antimycotic (1x), glutamine (4 mM), epidermal growth factor (20 ng/ml), and 0.5% fetal calf serum.7 cells/mL. Next, 100 μL of the PDX cell suspension was implanted into the mammary fat pad of 6- to 8-week old nude mice (Nu/Nu mice) following the protocol approved by the Institutional Animal Care and Use Committee at The University of Texas MD Anderson Cancer Center. Tumor growth in the mice was monitored once per week until tumors reached a volume of 500 mm3 to 800 mm3. Mice were monitored for signs of morbidity: failure to reach food and water; failure to thrive; inappropriate behavior such as lack of grooming, circling, etc.; a hunched posture, lateral recumbency, or distended abdomen; dyspnea; or weight loss >20%. Moribund animals were euthanized, and tumors and other tissues were removed for weighing and molecular marker studies.Animal experiments were conducted in full compliance with MDACC Institutional Animal Care and Utilization Committee (IACUC) policies and procedures (protocol ID: 00001305-RN00). Mice were housed under pathogen-free conditions and treated in accordance with NIH guidelines and standard rodent chow and water were available ad libitum. Tumors were implanted into NSG mice under Ketamine/Xylazine light anesthesia and Ketoprofen was used as an analgesic. At time of tumor harvest, mice were euthanized through CO2 inhalation followed by secondary exsanguination Previously archived, frozen IBC PDX tissue fragments (~100 mg) of the Bcx087 IBC PDX model were thawed and rinsed in PBS. Pieces of tumor tissue were mechanically fragmented with scissors prior to digestion in 3 mL of collagenase A (1 mg/mL solution in PBS) for 15 minutes at 37°C. Digested tissue samples were filtered through a 70-μm pore strainer, and the collagenase A was inactivated by addition of an equal amount of DMEM supplemented with 10% fetal calf serum. Samples were pelleted by centrifugation  and resuspended in 1 mL of red cell lysis buffer (Roche) for 2 minutes. Samples were centrifuged, and the cell pellet was resuspended in a 50:50 PBS:Matrigel solution to a final concentration of 2.0x10Prior to tumor harvest, mice were euthanized in a chamber containing CO2 from a cylinder as recommended by the American Veterinary Panel on Euthanasia . Every eEx vivo tumor tissue was generated by magnetic levitation using the Bio-AssemblerTM  as previously described [TM (nanoparticle assembly of iron oxide and iron nanoparticles cross-linked with poly-L-lysine) for about 18 to 24 h at a ratio of 1 μL of nanoshuttleTM to 150,000 cells to permit the binding of the iron nanoparticles to the surface of the cells. The nanoshuttle-coated cells were then placed in the Bio-Assembler system to bring the cells into a levitating aggregate  according to the manufacturer’s instructions.Total RNA isolated from the PDX tumors as well as from the in vitro transcription from double-strand cDNA templates containing T7 promoter. Fifteen micrograms of purified cRNA was reverse transcribed into second-cycle single-strand sense cDNA using second-cycle primers and dNTP mix with fixed dUTP/dTTP ratio in reaction mix. The single sense strand cDNA in second cycle was released from RNA/DNA hybrids by RNase H digestion. Then 5.5 μg of purified sense-strand cDNA was fragmented by uracil-DNA glycosylase and apurinic/apyrimidinic endonuclease 1 at the unnatural dUTP residues to break the DNA strand. The fragmented cDNA was labeled by terminal deoxynucleotidyl transferase using the Affymetrix proprietary DNA labeling reagent, which is covalently linked to biotin. Five micrograms of biotin-labeled sense-strand cDNA fragments in 200 μL of hybridization mix was loaded onto a human Clariom D array and hybridized to probes on the array in a GeneChip Hybridization Oven 645 (Affymetrix) for 16 h at 45°C with 60 rpm rotation. After hybridization, each array was washed and stained with streptavidin–phycoerythrin (Invitrogen), and the signal was further amplified with biotinylated anti-streptavidin antibody (Vector Laboratories) on the GeneChip Fluidics Station 450 (Affymetrix). Arrays were scanned with the GeneArray G7 scanner (Affymetrix) and quantified into CEL files with image and signal intensities.Whole transcriptome expression profiling was performed using an Affymetrix GeneChip human Clariom D array . Total RNA sample labeling and processing, GeneChip hybridization, and scanning were performed according to instructions in the Affymetrix user manual of GeneChip WT PLUS Reagent Kit–“Target Preparation for GeneChip Whole Transcript Expression Arrays (P/N 703174)”. Briefly, the first-strand cDNA was reverse synthesized from 100 ng of total RNA spiked in controls using a whole transcript plus kit, using primers containing a T7 promoter sequences . cRNAs were generated and amplified by http:///www.r-project.org/), and statistical significance was defined as p less than 0.05.The CEL files generated were analyzed through Transcriptome Analysis Console 4.0, which normalizes (and applies the log2 function to) array signals using a robust multiarray averaging algorithm. The Spearman's rank-order correlation test was applied to measure the strength of the association between gene expression in different arrays. The graphical representation of data and statistical analysis were performed in R (version 3.0.1) . Briefly, the slides were blocked for nonspecific binding and incubated with relevant antibodies for 1 hour at room temperature. The primary antibody was detected with the labeled streptavidin biotin plus peroxidase kit (DAKO). Slides were developed with DAB chromogen to reveal immunoreactivity, counterstained with hematoxylin, and covered with a glass coverslip. Antibodies used for immunohistochemistry were as follows: anti-E-Cadherin rabbit mAb (1:200) , anti-Vimentin XP rabbit mAb (1:200) , anti-Ki67 rabbit mAb (1:200) , and anti-pSmad2 (Ser465/467) rabbit mAb (1:200) .Median scores of viability across 3 replicates were normalized relative to the plate-level controls . The resulting values were then ranked from lowest to highest. Drugs ranking highest exhibited the strongest reduction in viability relative to the control. The statistical analysis for the in-vivo data described in the section “Value of the PDXEx model for predicting tumor response to drugs” is present in the paper by KURT et al. .ex vivo tumor tissue was determined on day 5 using the Cell Titer Glo Luminescent cell viability assay (Promega).Using robotics, 10 μL of the small molecules comprising the Anti-Cancer 386 Compound Library (Selleckchem) were dispensed into each well of a 96-well bio-printed PDXEx. Each well in the array contained 100 μL of medium. The small molecules were dispensed into 3 sets of plates for final concentrations of 1 μM, 0.5 μM, and 0.25 μM. Each drug concentration set was performed in triplicate. The bio-printed PDXEx was incubated with the drug concentrations for 5 days. Viability of the TM) and placed under a magnetic field (Bio-AssemblerTM), resulting in the cells’ been drawn together into a levitating mass , which has been shown to have a wide range of applications in tissue engineering [Having demonstrated that our IBC PDXEx model retained both the tissue architecture and expression profile of key biomarkers of the original PDX tumor, it was important to confirm that our model also retained the molecular signature of the original PDX tumor, which would allow our model to serve as a relevant preclinical platform for high-throughput drug screens. To compare the molecular signatures, we utilized magnetic 3D bio-printing  the cells in the model must retain the capacity to proliferate in culture to enable relatively rapid assessment of the anti-proliferative activity of novel therapeutic agents, and (ii) the method used to produce the model must produce robust and reproducible results with respect to similarity between the genetic signature of the original PDX tumor and the genetic signature of the PDXEx model. To assess the proliferative capacity of our PDXEx model, the viability of the bio-printed tissue was determined at 24 hours and 5 days after printing using the Cell Titer Glo Luminescent cell viability assay. The rationale for determining cell viability after 5 days of culture is that when the bio-printed PDXEx platform is used for drug screens, the bio-printed PDXEx will be grown in the presence of the drugs for 5 days before viability is measured. Our data clearly showed tissue proliferation during this 5-day period .To evaluate the robustness and reproducibility of the method used to generate the PDXEx model, we performed the method 3 times using PDX tumors harvested from 3 host mice, all of which had been implanted with the same original PDX tumor tissue. To ensure that the harvested PDX tumors had similar genetic signatures, we isolated purified RNA from these tumors and analyzed it using Clarion D human Affymetrix arrays comprising more than 6,765,500 probes derived from >134,700 characterized genes of both poly(A) and non-poly(A) mRNA, to provide complete and unbiased coverage of the transcriptome and profile differentially expressed genes. This complete gene set comprises coding genes, noncoding genes, microRNA, precursor micro RNA, pseudogenes, small RNA, and unassigned genes. The Spearman rank correlation was used to test the strength of the association in gene expression profiles between the 3 PDX tumors. We observed a highly significant genetic correlation (>90%) between the PDX tumors harvested from the 3 mice . A higheNext, in a similar manner, we determined the significance of the association in gene expression profiles between each PDX tumor and the resulting PDXEx model. Here too, a highly significant correlation (>87%) was observed between the gene expression profiles within each set , and a hWe next determined the proportions of genes that were similarly expressed and differentially expressed between each PDX tumor and the resulting PDXEx model. We first performed the analysis with the entire gene set  and thenTxn-1) and aspartate beta-hydroxylase (ASPH). Thioredoxin-1 [The lack of clearly defined molecular drivers for IBC contributes to the difficulty of identifying effective therapies to treat this disease. We too were unable to identify unique molecular drivers of the Bcx087 IBC PDXEx model. In edoxin-1 –69 and aedoxin-1  are highin vitro screen and the drug response profile identified in a previously described PDX mouse system [in vivo PDX mouse study but not in the PDXEx model. These findings confirmed the value of the PDXEx model in identifying effective tumor-specific therapies. The outcome of this study strengthens our belief that incorporating this PDXEx model within a PDX system to serve as a high-throughput screening platform will permit the identification of effective tumor-specific therapies in a time- and cost-efficient manner.To determine the value of our PDXEx model for predicting tumor response to drugs, we performed a pilot drug screen utilizing this platform and determined the similarity between the drug response profile identified in the e system . Both syUtilizing the Bcx087 bio-printed triple-negative IBC PDXEx platform, we performed a high-throughput small molecule screen with the Anti-Cancer 386 Compound Library (Selleckchem). The drug screen was performed in triplicate, and drug sensitivity as a percentage of the sensitivity of the solvent (DMSO)-treated cells was determined by measuring cellular proliferation using the Cell Titer Glo Luminescent cell viability assay (Promega). The drug screen was performed at drug concentrations of 1 μM, 0.5 μM, and 0.25 μM, and the platform was incubated with the drugs at 37°C for 5 days. Included in this screen were similar concentrations of the standard-of-care drugs doxorubicin and docetaxel , which sex vivo cultures of primary human tissue. These are preclinical models generated from tumor tissue slices and have been shown to recapitulate the tissue architecture of the original tumor and key features of the tumor microenvironment, including tumor heterogeneity [ex vivo model systems in a preclinical setting enables robust quantitative evaluation of clinically relevant anticancer therapies, drug resistance, and biomarkers and thus contributes significantly to identifying successful tumor-specific therapies and improving patients’ quality of life. Another benefit of ex vivo models is that by permitting recapitulation of inter-patient heterogeneity, they can be used to differentiate responders from non-responders to a treatment and thus aid in the selection of patients for clinical trials [Anticancer drugs developed utilizing 2-dimensional cell culture model systems have had minimal success when translated to the clinic . This faogeneity . The usel trials . All prel trials –79, incll trials . They teThe IBC PDXEx model that we describe appears to have potential as a solution to the current lack of suitable models for high-throughput identification of effective tumor-specific therapies for IBC. In this study, we clearly showed that i) the tissue architecture of our PDXEx model was very similar to that of the original tumor , ii) theOur PDXEx model is simple in design, taking only 11 days to reveal a tissue architecture similar to that of the original PDX tumor . In compReproducing the tumor microenvironment with a high degree of fidelity is a requirement for any cell culture system used for drug development because the tumor microenvironment not only influences the development of cancer but also has a strong influence on the tumor response to radiation and chemotherapy . The higin vivo study of the Bcx087 PDX model to validate the top drug candidates identified in our bio-printed PDXEx platform.The diagnosis of IBC relies largely on its clinical presentation; despite a characteristic phenotype, the molecular mechanisms underlying IBC are poorly understood . Analysiex vivo tumor tissue beyond 2 weeks. We do not consider this outcome a major impediment to use of our model since most ex vivo models derived from slices of tumor maintain cell viability over culture periods of at least 1 week [ex vivo models, use of our PDXEx model as a screening platform requires good availability of biopsy tissue, which could be a limitation in patients with IBC. However, the use of PDX tumor tissue harvested from mice will overcome this limitation, affording us the ability to identify agents with a high likelihood of clinical efficacy before clinical trials or even animal studies. This outcome would undoubtedly improve the efficiency of translating preclinical research to clinical practice. Incorporating ex vivo culture of IBC tumors into the development pipeline could help us to achieve this outcome, and this approach has been increasingly used to investigate potential prostate cancer therapeutics [Ex vivo models such as organoid models have been generated from specific cells within tumors with characteristics similar to those of the original tumor [Although our PDXEx model has potential clinical benefits, it also has one minor drawback. Despite many attempts, we were unable to prolong viability of our t 1 week . Similarapeutics  using tial tumor . The advex vivo cultures can enable quantitative evaluation of multiple drugs. We postulate that if a drug cannot elicit an anti-proliferative or proapoptotic response in a high proportion of patient tumors in an ex vivo system, that drug is unlikely to be effective in the clinics. Here, we show the importance of ex vivo cultures enabling the evaluation of drug efficacy using a high-throughput strategy. We are confident that our preclinical ex vivo tumor tissue array platform will ultimately be confirmed as a clinically relevant predictor of tumor response to drugs and improve understanding of the biology of IBC.Further work is needed to determine if our preclinical PDXEx platform will serve as a clinically relevant predictor of tumor response to drugs and improve understanding of the biology of IBC. We have shown in the work described here the importance that


Microsatellite instability (MSI) marks distinct subsets of tumors in many cancer types and is caused by mutations in genes required for mismatch repair. A recent report analyses the molecular foundations of MSI-positive colorectal cancers and reveals substantial molecular heterogeneity, which might have consequences for the potential use of immunotherapy in MSI-positive cancers.See related research by Sveen et al. 10.1186/s13073-017-0434-0 Hereditary patterns of colon cancer were recognized as early as the 1960s by Henry Lynch. He described Lynch Syndrome, or hereditary non-polyposis colorectal cancer, which is characterized by an autosomal-dominant inheritance pattern and a marked propensity to develop cancers, including colorectal cancer. However, the mechanistic understanding that the tumors of these patients had high numbers of somatic mutations in regions of short tandem repeats or microsatellites and the identification of the causative genetic alterations came decades later [Microsatellite instability (MSI), either due to inherited germline mutations of mismatch repair (MMR) genes or epigenetic inactivation of these genes, is found in approximately 15% of stage II to III colorectal cancers (CRCs) . MMR defThe prognostic significance of MSI in CRC has been demonstrated in numerous studies. Decreased rates of recurrence and metastasis, and increased survival occurs in patients whose tumors harbor MMR deficiency in comparison with MMR-proficient tumors . In addiWhereas typical non-MSI tumors may have a hundred or so mutations in the exome, MSI-positive CRCs characteristically have thousands of mutations throughout the coding regions of tumor cells. These tumors are also often characterized by increased numbers of tumor-infiltrating lymphocytes (TILs), including cytotoxic T cells—thought to be a response to the increased number of neoantigens produced by the tumor resulting from the high mutational load. The neoantigens are presented in the context of major histocompatibility complexes (MHCs) and are recognized by the host immune system as foreign . In ordeGenome Medicine, Sveen and colleagues [CRTC1 (CREB-regulated transcription coactivator 1), CCND1/BCL1 (G1/S-specific cyclin-D1), PTCH1 (protein patched homolog 1), and JAK1 (tyrosine-protein kinase JAK1), although many were subclonal , consistent with significant heterogeneity within the tumor. JAK1 loss-of-function mutations were found in several cohorts, with a prevalence in MSI-positive CRCs of 20%, although mutations were primarily heterozygous. Interestingly, mutated tumors were associated with upregulation of genes associated with resistance to anti-PD1 treatment. However, the heterozygous nature of these mutations and positive association with disease outcomes seen in this study are inconsistent with previous reports, including a recent study reporting association of JAK1 mutations with resistance to PD-1 blockade in a small cohort of patients [JAK1 mutations might impart a better prognosis in the absence of immune-checkpoint blockade therapy, homozygous JAK1 mutation results in a resistance to antibodies against PD1 in patients treated with immunotherapy. Further studies are thus required to resolve the ultimate biological effect of these mutations.In this issue of lleagues  analyzedpatients . It is pJAK1 mutations and the CMS1 subtype were both significantly associated with improved survival.As expected, Sveen and colleagues observed increased neoantigen load accompanying increasing mutational load even within these MSI-positive tumors. Immune infiltration, as measured by gene expression, was associated with the CMS1 gene-expression subtype but was not associated with increased mutational burden. Here, it is possible that specific antigens are present in this group of tumors or they have a microenvironment that is more permissive to T-cell infiltration. These results represent the largest analysis devoted to MSI-positive CRCs reported to date and offer key insights into our understanding of MSI and potential determinants of the response to immunotherapies. An important conclusion of this study is that not all MSI-positive tumors are the same, as there exist markedly different immunophenotypes and clinical behaviors within this subgroup of tumors. Considering that mutational load and microsatellite instability are soon to be used in the selection and/or prioritization of patients to receive immune-checkpoint inhibitors, a greater understanding of the heterogeneity within hypermutated tumor subsets and operative drivers of resistance is crucial for their success as clinical biomarkers. This study provides a nice window into the heterogeneity that must be accounted for as researchers optimize the use of immunotherapeutics for the treatment of human cancers.


Ductile cutting are most widely used in fabricating high-quality optical glass components to achieve crack-free surfaces. For ultra-precision machining of brittle glass materials, critical undeformed chip thickness (CUCT) commonly plays a pivotal role in determining the transition point from ductile cutting to brittle cutting. In this research, cutting characteristics in nanometric cutting of BK7 and fused silica glasses, including machined surface morphology, surface roughness, cutting force and specific cutting energy, were investigated with nanometric plunge-cutting experiments. The same cutting speed of 300 mm/min was used in the experiments with single-crystal diamond tool. CUCT was determined according to the mentioned cutting characteristics. The results revealed that 320 nm was found as the CUCT in BK7 cutting and 50 nm was determined as the size effect of undeformed chip thickness. A high-quality machined surface could be obtained with the undeformed chip thickness between 50 and 320 nm at ductile cutting stage. Moreover, no CUCT was identified in fused silica cutting with the current cutting conditions, and brittle-fracture mechanism was confirmed as the predominant chip-separation mode throughout the nanometric cutting operation. Precision optical components, made of hard-brittle materials such as ceramics and glasses, always require high-quality surface with good edge and crack-free zone. However, fabricating these components still is the most difficult and challenging task for modern manufacturing sectors due to the key obstacle to achieve acceptable machining quality. In spite of this, the demands for microfeatures in these materials have been increasing for a number of miniature-product applications such as MEMS  device packaging, mini-vision systems and microelectronic packaging . Unfortuet al. [et al. [et al. [Boroscilate crown glass (BK7) and silicon dioxide glass  are two common optical materials for fabricating of high-quality optical components due to their excellent properties, such as good scratch resistance, bubble-free, and high optical transmission in the visible range ,9. The met al.  investig [et al.  carried  [et al.  establisIn this study, nanometric plunge-cutting experiments were performed on an ultra-high-precision CNC machining center. Cutting characteristics were investigated for both BK7 and fused silica according to the variation of surface texture generation with increasing undeformed chip thickness. In addition, several aspects including surface roughness, cutting-force generation and specific cutting energy were precisely investigated aiming to clarify the ductile-brittle transition point and the material removal regime during ductile and brittle cutting.Plunge-cutting experiments were carried out on a 5-axis ultra-high-precision CNC  machining center  with a position detection and motion control by minimum unit of 0.07 nm, as shown in ® software. The optical profilometry was also used to measure the nanometric surface roughness along the bottom of cutting groove in the feed direction. Scanning electron microscope (SEM)  was also used to characterize the machined surface texture, such as fractures and micro-chips. Cutting forces were measured by a piezoelectric dynamometer . The onset of brittle fracture was carefully observed through 3D surface morphologies and cross-sections of the groove, which were detected by an optical profilometry  with MetroProet al. [et al. [As shown in et al. . With re [et al.  proposed [et al. . There acF is the feed cutting force, and A is the cross-sectional area of the groove, as shown in A can be calculated as:rε is the nose radius of the SCD tool, and d is the depth of the groove .Specific cutting energy represents the energy consumed in removing a unit volume of materials. It exhibits strong relation with the cutting load and also can be used to distinguish the mode of material removal . The speet al. [cd is CUCT, E is the Young’s modulus, H is the nano-hardness, KIC is the fracture toughness of mode I, and β is a dimensionless material constant determined by the cutting conditions. β was estimated as 0.15 for brittle materials derived by Bifano [The cutting force and cross-sectional area both depend significantly on the undeformed chip thickness. It is believed that there always exists a critical value when an initial crack appears at the ductile-brittle transition point, which can be precisely observed by Zygo optical profilometry in this study. Based on Griffith fracture propagation criterion, Bifano et al.  proposedy Bifano  in preciThe measured 3D surface morphologies and cross-sections of the cutting groove for BK7 are shown in As presented above, BK7 and fused silica show distinct removal regimes in the nanometric plunge-cutting process. Typically three stages of ductile, ductile-brittle transition and brittle could be observed for BK7 cutting, while the fused silica cutting only exhibited the brittle stage with numerous brittle fractures of materials. Such phenomena can also be confirmed from the following SEM micrographs of the cutting grooves, as shown in At the beginning of ductile cutting, there are many sliding and plowing traces on the machined surface of the cutting groove, as shown in As presented above, CUCT of BK7 was observed as 320 nm and no CUCT was found for fused silica in the plunge-cut tests. The experimental results are different from the theoretical CUCT calculated with Equation (3) in Surface finish of the cutting groove was investigated to characterize the surface generation regime. Surface roughness was measured along the bottom of groove. Low surface roughness value always signifies a smooth surface with minimum cracks or fractures. The material removal mechanism for BK7 cutting could also be revealed by specific cutting energy. As shown in A of the cutting groove for fused silica cannot be calculated by Equation (2). So the specific cutting energy was not discussed in this section. Further research is necessary to find the CUCT and to get the specific cutting energy for fused silica by using SCD tools with effective negative rake angle.It was different from BK7 cutting that brittle fractures were main material removal regime for fused silica cutting. Irregular cross-sections of the cutting groove were always caused, as shown in (1)There existed a CUCT of 320 nm in nanometric cutting of BK7, which experienced ductile, ductile-brittle transition and brittle cutting stage successively with increasing undeformed chip thickness. Only brittle cutting was found for fused silica with the same cutting conditions when undeformed chip thickness was above 30 nm, and brittle fractures existed for its high hardness and low fracture toughness compared with BK7.(2)Low surface roughness value could be obtained in ductile cutting and ductile-brittle transition cutting during BK7 nanometric plunge-cutting test. Brittle cutting process was confirmed to cause the significant increase of machined surface roughness irrespective of BK7 or fused silica. The key reason was attributed to the large amount of cracks and fractures on the machined surface.(3)The feed cutting force almost increased linearly with elevated undeformed chip thickness for BK7, while the feed cutting force for fused silica fluctuates violently and the amplitude of fluctuation increases when undeformed chip thickness increased. The phenomena were mainly due to the large brittle fractures that led to the high cutting force and fluctuation.(4)Size effect was found with undeformed chip thickness below 50 nm in ductile cutting of BK7. The appropriate undeformed chip thickness between 50 and 320 nm was expected to get a good surface integrity, when precision machining of BK7 with the similar cutting conditions as used in this research.An experimental investigation with nanometric plunge-cutting test has been carried out on two hard-brittle materials of BK7 and fused silica. In the experiments, SCD tool with 0° rake angle was used under the same cutting speed of 300 mm/min. Cutting characteristics was studied with special concentration on the material-removal regime based on ductile-brittle transition. Based on the research, the following conclusions can be drawn:


A novel method of protein labeling uses the highly dynamic reversible association of a cell-permeable fluorogenic dye and lipocalin Blc mutants. We present protein-PAINT – the implementation of the general principles of PAINT  for live-cell protein labeling. Our method employs the specific binding of cell-permeable fluorogenic dyes to genetically encoded protein tags. We engineered three mutants of the bacterial lipocalin Blc that possess different affinities to a fluorogenic dye and exhibit a strong increase in fluorescence intensity upon binding. This allows for rapid labeling and washout of intracellular targets on a time scale from seconds to a few minutes. We demonstrate an order of magnitude higher photostability of the fluorescence signal in comparison with spectrally similar fluorescent proteins. Protein-PAINT ensures prolonged super-resolution fluorescence microscopy of living cells in both single molecule detection and stimulated emission depletion regimes. Fluorescence labeling of target proteins in live cells provides opportunities to visualize their intracellular distribution, interactions, and dynamics. Green Fluorescent Protein (GFP) and GFP-like proteins are most often used for this purpose as fully genetically encoded labels.Halorhodospira halophila to reversibly bind fluorogenic rhodanine derivatives. However, both labeling systems do not provide an increase in photostability, which could be expected from the exchange of the protein-bound and free dye molecules from solution.These steps could be avoided by using fluorogenic dyes – those that are non-fluorescent in solvents but become fluorescent in a complex with the molecule of interest.e.g. antibody). However, even the latest-generationConstant exchange of the dye provides a method for efficient single molecule super-resolution microscopy, since the binding is detected as a flash of fluorescence signal. The original demonstration of this principle on membranes with the Points Accumulation for Imaging in Nanoscale Topography (PAINT) techniqueHere, we created novel fluorogen–protein pairs for live-cell protein labeling and validated them in widefield, confocal and super-resolution microscopy applications. With these pairs we demonstrate the protein-PAINT principle: the combination of the genetically encoded protein tag and the rapid exchange of the fluorogenic dye allows for on-demand fluorescent labeling of target proteins in living cells and is compatible with various super-resolution techniques.GFP chromophore analogs have been previously reported to be fluorogenic with RNA aptamersE. coli without intramolecular disulphide bonds.27Application of a chromophore-containing solution to cells expressing host proteins or RNA allows for the fast formation of fluorescent complexes, but also – if the dye is not bound to the host too tightly – for efficient exchange of dye molecules with the pool of free molecules. Therefore, photobleached dye molecules can be quickly exchanged for the fluorescent ones. Taking this as a starting point, we designed and synthesized the library of GFP chromophore analogs, GFP-like aminated chromophores, and conformationally locked compoundsin silico mutagenesis of amino acids within the ligand-binding pocket of Blc , DiB2 (Kd = 4 μM) and DiB3 (Kd = 9 μM) (M739 showed blue- (for DiB1 and DiB2) or red-shifted (for DiB3) absorption spectra with similar extinction coefficients in the range of 43 000–51 000 M–1 cm–1   and S1†. Fig. S3,. CompareDiB1 and DiB2, the effective wavelength-dependent fluorescence increase of the complex was more than 50-fold . We found that the two yellow-colored pairs (DiB1 and DiB2) performed well in both widefield and confocal fluorescence microscopy. The target intracellular structures of the live cells demonstrated bright fluorescence immediately upon addition of the fluorogen M739 at concentrations of 0.1–1 μM  histone H2B in living cells with the photostability of spectrally similar fluorescent proteins. We found that the protein–fluorogen pairs with higher Kd values (DiB2 and DiB3) exhibited an order of magnitude higher photostability than the corresponding fluorescent proteins  in live HeLa Kyoto cells with an EM-CCD camera, allowing for the detection of single molecule emitters  indicate the possible link between the residence time (determined by koff) of the fluorogen within the complex and the irreversible photobleaching. Indeed, photobleaching of GFP-like proteins occurs through multistep photochemical reactions of the chromophore, amino acids of the protein host and external compounds.koff was also reported for some FAP variants.DiBs could be used for single molecule localization microscopy (DiB2 and DiB3) exhibited the full advantages of the dynamic dye interchange. Thus, the protein-PAINT labeling method with DiB2 and DiB3 allows for the prolonged super-resolution imaging of live cells with the density of single-molecule fluorescence bursts controlled by the concentration of the dye in the imaging solution.Addition of the compound nditions , in acconditions . The obscroscopy  and S6†,DiBs is also beneficial for other light-intensive super-resolution modalities, such as stimulated emission depletion (STED). We observed a dramatic increase in the photostability (in comparison with a fluorescent protein) of DiB1 and DiB2 in STED mode on the same scale .Supplementary movieClick here for additional data file.Supplementary movieClick here for additional data file.Supplementary movieClick here for additional data file.Supplementary informationClick here for additional data file.


The increasing number of people with dementia and cognitive impairments makes it essential to detect and prevent modifiable risk factors of dementia. This study focuses on type 2 diabetes mellitus, especially on undiagnosed cases and their increased risk of cognitive impairment. Furthermore, the potential of physical activity and social integration to moderate the relation between diabetes and cognitive impairment is assessed.We used cross-sectional data from 1299 participants of the Berlin Aging Study II (BASE-II) aged between 60 to 84 years and performed logistic regression models to analyze the association of diabetes status, physical activity, and cohabitation status with poor cognitive performance. Cognitive performance was measured with the Consortium to Establish a Registry for Alzheimer's Disease (CERAD)-Plus test battery.Undiagnosed diabetes (odds ratio (OR) = 2.12, p = 0.031), physical inactivity  and non-cohabiting  were associated with an increased likelihood of poor cognitive performance. The highest odds were observed in participants who suffered from undiagnosed or insulin-dependent diabetes and, in addition, were inactive  or lived alone . Physical activity and cohabiting appeared to be beneficial.Physical activity and cohabitation status moderate the link between diabetes mellitus and cognitive performance. Special attention should be paid to undiagnosed and insulin-dependent diabetes cases, which have a particularly high risk of poor cognitive performance. Cognitive impairments and dementia are among the leading risk factors for disability and death –3. The iAnother important aspect of a diabetes related life style factor is regular physical activity. Numerous studies have proven the beneficial effects of physical activity on cognition –23. A meIn addition to physical activity, the cohabitation status and levels of social integration also affect the development of cognitive decline , 25. PeoIn the present study we analyze whether the presence of diabetes mellitus, physical activity, and cohabitation status are correlated with cognitive performance for members of a community-dwelling elderly population. We differentiate between non-diabetics, diabetics treated with oral anti-diabetic medications (ADM), diabetics treated with insulin, untreated diabetics, and undiagnosed diabetics. We hypothesize that diabetics with effective glycemic control have comparable risks of poor cognitive performance when compared to non-diabetics, whereas insulin-dependent diabetics show increased risks of cognitive impairment as they are probably in a later and more severe stage of the disease . In a loThe Berlin Aging Study II (BASE-II) is an ongoing joint project of various disciplines involving several institutions. The population-based sample of community-dwelling participants, living in the greater metropolitan area of Berlin, Germany, covers numerous ageing-relevant variables . The samFor our analyses we used data of the older subsample and included only those individuals with complete information on diabetes status, physical activity, cohabitation status, and the neuropsychological test battery, resulting in an analytical sample of 1299 participants.To measure cognitive performance we used the German version of the neuropsychological test battery CERAD-Plus (Consortium to Establish a Registry for Alzheimer's Disease) –33. The  effects , 35. In Study participants were defined as diabetic if one of the following criteria was fulfilled: (1) Subjects listed a diabetes diagnosis in the questionnaire; (2) Intake of an oral anti-diabetic drug or insulin; (3) Glycated haemoglobin (HbA1c) levels over 6.5%; (4) Fasting plasma glucose (FPG) over 126 mg/dL; (5) 2-hour glucose level over 200 mg/dL . The 2-hPhysical activity was assessed with the Rapid Assessment of Physical Activity (RAPA) questionnaire , 38. We The cohabitation status was assessed with the question “How do you live?”. We distinguished between people who lived together with a partner or a relative  and subjects living alone.2 vs. 30 kg/m2 or more) [The following covariates were entered into the statistical analyses: Sex; age in years as metric variable (ranging from 60 to 84); education ; body mass index (less than 30 kg/mor more) ; currentor more) ); and se2 tests for categorical variables. We performed univariate logistic regression models with all used covariates and three multivariate logistic regression models for calculating the odds ratios (OR) of poor cognitive performance. With the exception of age, all independent variables were included as dummy variables. We extended our models by interaction terms in order to test for moderator effects of physical activity and cohabitation status with diabetes mellitus. All analyses were performed using STATA 12.1.We compared the characteristics of the participants by their diabetes status and used one-way ANOVA for continuous variables and χUnivariate logistic regression analysis revealed that diabetics compared to non-diabetics had a statistically increased odds ratio (OR) of 1.82 (p = 0.001) of poor cognitive performance . Table 4Models with interaction terms revealed that the likelihood of poor overall cognitive performance was particularly high if people suffered from undiagnosed diabetes  or were treated with insulin  and were also inactive . Also inIn the current study undiagnosed diabetics without effective glycemic control were shown to have the highest risks of poor cognitive performance, which is in line with a previous study by Xu and colleagues . A consiAlong with the status of diabetes, life style variables had an influence on the likelihood of poor cognitive performance. People who were inactive and living alone have significantly elevated risks, which is in line previous studies –26. FratThe combination of undiagnosed or insulin-dependent diabetes and inactivity or non-cohabitation was particularly deleterious. However, if participants were regularly physically active or lived together with a partner or relative, the risks of poor cognitive performance did not differ from those of active or cohabiting non-diabetics. This result was independent of the treatment status of the diabetics. Still, the enhanced risk due to undiagnosed or insulin-dependent diabetes could partly be compensated for by physical activity and social integration. Both factors most likely counteract the harmful effects of diabetes and insufficient glycemic control. This finding is in line with a recent a study indicating that moderate-to-high intensity aerobic exercise may have a disease-modifying effect in terms of reduced levels of tau proteins in cerebrospinal fluid, increases in blood flow in the brain, and improvements of executive functions in elderly people with MCI and prediabetes .The present study does have some limitations. First, the cross-sectional design does not allow us to draw causal conclusions. There is always the possibility of reverse causation, meaning that cognitive impairment is the cause of an unhealthy life style which leads to diabetes or inactivity. Second, social integration was assessed in conjunction with the cohabitation status. Living alone does not necessarily mean that people are not socially integrated. Nevertheless, cohabiting is one of the main components of social interaction  and we wThe strength of this study is the large number of cases with available information on their cognitive status measured with a total score of the CERAD-Plus test battery, which is superior to the MMSE in detecting MCI , 35. TheSeveral studies have indicated a decreasing trend of dementia prevalence and incidence –50, primA healthy life style and goal-directed detection and treatment of diabetes might contribute to the continuation of decreasing incidence and prevalence of dementia. Further research is needed to analyze to what extent these factors influence the development of cognitive impairments and dementia.S1 TableSource: BASE-II.(DOCX)Click here for additional data file.S2 TableSource: BASE-II.(DOCX)Click here for additional data file.S3 TableSource: BASE-II.(DOCX)Click here for additional data file.


Sensitive detection of proteins by X-ray fluorescence microspectroscopy using the Re core of a single core multimodal probe for imaging. Z ≥ 14) in cells or biological samples. In this context, similar tools to those developed for fluorescence microscopy are desired, including chemical probes or tags. In this work, we study rhenium complexes as a convenient and sensitive probe for X-ray fluorescence microspectroscopy. We demonstrate their ability to label and sense exogenously incubated or endogenous proteins inside cells.Bio-imaging techniques alternative to fluorescence microscopy are gaining increasing interest as complementary tools to visualize and analyze biological systems. Among them, X-ray fluorescence microspectroscopy provides information on the local content and distribution of heavy elements ( However, fluorescence techniques suffer from some drawbacks such as photobleaching. Quantum yields are dependent on the environment, with large variations upon polarity or possible quenching at high concentrations.Bio-imaging enables the direct visualization of labeled biomolecules of interest, offering valuable insights into biological processes. Fluorescence microscopy is widely used and offers numerous advantages, including good spatial resolution  tricarbonyl complexes as multimodal probes for fluorescence microscopy and infrared microspectroscopy.Accessibility to new probes for this emerging technique would bolster its use for further investigation of biological mechanisms. Studies have been performed in this direction with the development of particles, quantum dots, polymers containing lanthanide and carbon nanotubes.i) as a tag for SXRF, we wanted to assess whether low-abundant proteins could be labeled with SCoMPIs and mapped by SXRF-MS in a cellular environment. To do so, we explored two different approaches: (1) in vitro labeling of proteins that can be internalized in cells and (2) labeling of membrane-bound endogenous proteins in cells (In order to test the efficiency of Re(in cells .Engrailed homeodomain. Homeodomains (HD) are domains of the highly conserved homeoproteins that are able to penetrate cells.Engrailed homeodomain, in particular, has been used as a model to study some of the unconventional translocation mechanisms of homeodomains.Engrailed 2 homeodomain itself, and (2) NLS-HD displays an extended sequence with a putative Nuclear Localisation Signal (NLS) sequence. Neither HD nor NLS-HD bear a cysteine residue in their native sequence and for both constructions, a single cysteine was introduced at the N-terminus, for site-specific labeling of the protein through thiol–maleimide coupling (see ESIi)-catalyzed azide–alkyne cycloaddition (CuAAC) “click” chemistry between 2-ethynylpyridine and an azide with a relevant linker/function (here a Boc-protected amine). The reaction of this ligand with the Re(CO)5Cl precursor resulted in the formation of a Re(i) tricarbonyl complex. Finally, the amine was deprotected and reacted with an activated ester linked to the maleimide moiety  or NLS-HD (Re-NLS-HD), washed and chemically fixed using an aqueous solution of paraformaldehyde (PFA). The chemical fixation is well suited in this case since the probe is covalently grafted to the proteins, which are immobilized by PFA fixation. Consequently, we assumed there is little risk of re-localization upon fixation.A five-fold excess of the SCoMPI functionalized with a maleimide was used to ensure a complete reaction and both constructs could be efficiently labeled I see ESI. FluoresI see ESI showing et al. manuscript in preparation).None of the SCoMPI-labeled proteins could be detected by SR-FTIR microspectroscopy nor discriminated from the background signal of cells by fluorescence microscopy. But, as shown in Encouraged by these findings, we wanted to investigate whether endogenous proteins could also be detected by SXRF-MS using Re tags. We decided to use the “Traceless Affinity-Labeling” strategy developed by Hamachi's group, as it enables the modification of endogenous proteins without prior engineering of the protein of interest (POI) or major loss of protein function .39–41Traceless labeling relies on the use of a ligand (inhibitor) of the POI, linked to a tag by a cleavable linker. Upon binding to the protein through ligand recognition, the cleavable linker is brought in close proximity to nucleophilic residues on the POI, which leads to the covalent binding of the tag to the protein. The so-called “Ligand-Directed Acyl Imidazole” (LDAI) chemistry was shown to have interesting features with regards to the reactivity and stability of the reagent in biological media.47ca. 80%) was obtained within 2 h. In the presence of EZA, no labeling was observed. This confirms that the labeling is driven by the recognition of the ligand.The convergent synthesis of LDAI reagents consisted of the synthesis of ligand-imidazole moieties and of a NHS-activated carbonate analog of the probe, which were then coupled, leading to the LDAI reagent (see ESI and Schemes S2–S43N4 slides, placed under hypoxic conditions for 24 h, and then incubated with 10 μM of LDAI-SCoMPI. After washing of the cells to remove the unreacted LDAI-SCoMPI, cells were fixed with PFA and mapped using XRF imaging. This technique provides a projected image (Carbonic anhydrases are overexpressed under hypoxic conditions. A549 cells were grown on Sied image . Interesed image . This coIn conclusion, the results presented herein showed that it was possible to label and map by X-ray fluorescence an exogenous protein in CHO cells and membrane bound proteins in A549 cells, which establishes the high sensitivity of Re-based probes for X-ray imaging. This proof of concept offers new perspectives in XRF imaging, for which so far no specific graftable tags were available. This third modality of SCoMPI opens new opportunities for correlative imaging.There are no conflicts to declare.Supplementary informationClick here for additional data file.


The development of single-cell RNA sequencing has enabled profound discoveries in biology, ranging from the dissection of the composition of complex tissues to the identification of novel cell types and dynamics in some specialized cellular environments. However, the large-scale generation of single-cell RNA-seq (scRNA-seq) data collected at multiple time points remains a challenge to effective measurement gene expression patterns in transcriptome analysis.We present an algorithm based on the Dynamic Time Warping score (DTWscore) combined with time-series data, that enables the detection of gene expression changes across scRNA-seq samples and recovery of potential cell types from complex mixtures of multiple cell types.https://github.com/xiaoxiaoxier/DTWscore.The DTWscore successfully classify cells of different types with the most highly variable genes from time-series scRNA-seq data. The study was confined to methods that are implemented and available within the R framework. Sample datasets and R packages are available at The online version of this article (doi:10.1186/s12859-017-1647-3) contains supplementary material, which is available to authorized users. Methodological advances provide transcriptomic information on dozens of individual cells in a single-cell sequencing project –3 to stuk−nearest neighbor graphs without dimensional reduction. Critically, dimensional reduction does not make full use of the rich information provided by scRNA-seq time-series data. However, the existing methods may overlook un-synchronization over the entire time series. It is a challenging problem to provide the approaches to identify the set of genes from distinct cells that are differentially expressed over time. Moreover, estimating at which time periods the transcriptional heterogeneity with different cell types is present can provide additional insight into temporal gene functions.Among others, one key objective is to define the sets of genes that best discriminate transcriptional differences by inferring the heterogeneity of cells’ unsynchronized evolution . This stIn this article, we present an algorithm based on the Dynamic Time Warping score (DTWscore)  that is For single-cell RNA-seq data, the gene expression level of some fixed time points become more easily obtainable than traditional bulk RNA-seq data . A commoBriefly, the DTWscore focuses on detecting the cell-to-cell heterogeneity among time-period scRNA-seq data and highlights the highly divergent genes that are used to define potential cell types. The input of the DTWscore is a matrix of time-series gene expression data. The rows of the matrix stand for individual genes, and the columns represent the gene expression profiles of different cells from discrete time points. The method is performed on both simulated and real datasets. In particular, if a gene expression level between different time periods is quantified through the same process function, we consider genes of this type to show non-heterogeneity across cells, while the remaining genes are deemed as highly variable genes between time series data for further analysis. A graphical representation of our method pipeline is displayed in Fig. t to simulate gene expression patterns with four different ‘biological processes’(see Methods for details). If the gene expression patterns are tracked during the unfolding of a biological process, the process can be conceived as some specific functions over time. Four typical trajectories of gene expression are simulated graphically  and f3(t), while the red curve represents condition 2 simulated by the biological functions f2(t) and f4(t).In the simulation, two groups of scRNA seq data with time are generated as follows. Group one (non-heterogenerous genes): the gene expression matrix at multiple time points is generated by the same function shown in Fig. Mclust function from R package mclust [In this section, DTWscore is applied to the recently published data from human skeletal muscle myoblasts  which wee mclust , 25 to pIn order to assess the performance of DTWscore in relation to other approaches, we run Monocle and SLICER on the HSMM data and compared the classification results from all the three approaches.Monocle uses independent component analysis (ICA) to reduce the dimensionality of the expression data before clustering the cells. Monocle also provides algorithms on unsupervised cell clustering and semi-supervised cell clustering with known marker genes. Figure k-nearest neighbor graph shows the clustering using SLICER branching analysis. It appears that SLICER benching analysis suggest that cells should fall on many different branches which maybe more than the real number of cell types. Obviousily SLICER is capable of detecting types of features but sometimes it will overfit. However, DTWscore is a model-based method to infer the potential cell types which is more flexible for diverse datasets.Because SLICER can infer highly nonlinear trajectories and determine the location and number of branches and loops, the cells fall on more different branches. Figure We stress that our method is different from the approach that detects cell clusters and expression differences, such as those described previously , 15, 20,The approach is limited in that only classification of cell types are feasible. A generalized DTW algorithm used for the analysis will make analyses of more than three to four cells over time possible; work in that direction is underway. Finally, we note that, while the differentially expressed genes identified by the DTWscore may prove useful in downstream analysis and cellular branches and trajectories inference, extensions in this direction are also underway.To date, a large amount of available high-throughput data has been measured at a single time point . Time-seOur analysis of scRNA-seq time-series gene expression datasets increased the ability to study various cellular mechanism over time. First, in HSMM cells, we identified highly significantly differentially expressed genes with time-series data, indicating that the genes are marked for use in the following clustering. The expression of these genes possibly arose from the un-synchronized time-series scRNA-seq experiments. Second, given the various biological processes, the DTWscore for each gene was calculated using our pipeline. By combining the method to set thresholds, quantitative analysis has enabled the direct separation of heterogeneous and non-heterogeneous genes. The DTWscore can manage uneven and sparsely sampled time series gene expression data without need for prior assumptions about the evenness or density of the time-series data. Moreover, all pairs of cells are calculated by DTWscore, a procedure that could result in the stability of finding important highly variable genes. Finally, the DTWscore could successfully identify the potential cell types from bunch of scRNA-seq data.Regarding computational future directions, recovering the genes’ heterogeneity over time in individual cells is only a fist step in understanding the complex dynamic processes that drive changes in gene expression. Most scRNA-seq data sets consist of hundreds (and sometimes thousands) of cells that have recently allowed parallel sequencing of substantially larger numbers of cells in an effective manner, which brings additional challenges to the statistical analysis of scRNA-seq data sets . We expect that developing unified computational methods with time-series single cell gene expression data will yield more biological insights. Inferring the potential types and states of individual cells is thus a useful tool for studying cell differentiation and govern a much wider array of biological processes.ci∼N and εi∼N. For the actual values of t, we used the sequence range from 0 to 10∗π of 314 values. We considered 1000 genes from two types, highly variable genes and non-heterogeneous genes; and two cells from two time series. For each cell, we varied the interval of each different time points, and the corresponding DTWscore is calculated for each gene.We performed our pipeline on both synthetic time series data and real temporal gene expression data downloaded from (GEO). The real time-series scRNA-seq data were obtained from GSE52529 . The datThe DTWscore pipeline contains four steps Fig. .The first step in single-cell RNA-Seq analysis is identifying poor-quality libraries. Most single-cell workflows will include at least some libraries made from dead cells or empty wells in a plate. The expression level of each gene was represented by FPKM values. DTWscore hold the genes expressed in at least 80 percent of the total cells in the data set. Genes that were non-expressed in more than 80 percent of the total cells were excluded, leaving the remaining genes for further analysis. Consequently, thousands of genes could be reduced to hundreds for further analysis.np temporal gene expression values. Let i of jth time points in the pth  cells. Briefly, if the expression levels of some gene i are tracked during the unfolding of a biological process, the process can be conceived as tracing out a trajectory over time. We consider the two temporal gene expression in two cells as two time series: f is defined between any pair of elements xm and xn, with some type of distance: The DTWscore is calculated based on the FPKM gene expression levels. The dynamic time warping technique  is used ϕ such that Note that the most common choice is to assume the Euclidean distance, different definitions  may be ing path . ConsequThe DTW algorithm makes use of dynamic programming and works by keeping track of the cost of the best path at each point in the grid: Consequently, amed dtw , which pDi,for each gene is obtained. Suppose Model-based threshold to identify highly variable genes will change significantly among various types of datasets. As the variabilities are high in scRNA-seq time-series data, a fixed threshold for the DTWscore is less effective in many settings. Flexible thresholds for the DTWscore are necessary, allowing the test of variabilities in response to a numerically estimated trend in the predictors, alleviating the burden of specifying their distribution. We utilized the distribution model to identify the specific gene sets for further analysis. We briefly summarize the main insight. As noted above, the empirical distribution of the DTWscore from all time-course datasets falls into normal distribution Fig. . ProbabiWe can classify cells as follows. First, to cluster the cells, we choose the gene with the highest DTWscore and make full use of its expression values of all the time points. Next procedure requires R package mclust which provides Gaussian finite mixture model fitted by EM algorithm . The rocAdditional file 1Figure S1. DTWscore identifies heterogeneous genes and non-heterogeneous genes from the synthetic data (condition 3). (PDF 122 kb)Additional file 2Figure S2. DTWscore identifies heterogeneous genes and non-heterogeneous genes from the synthetic data (condition 4). (PDF 122 kb)Additional file 3Figure S3. DTWscore identifies heterogeneous genes and non-heterogeneous genes from the synthetic data (condition 5). (PDF 122 kb)Additional file 4Figure S4. DTWscore identifies heterogeneous genes and non-heterogeneous genes from the synthetic data (condition 6). (PDF 122 kb)Additional file 5Figure S5. Model-based clustering of HSMM dataset by any two highly variable genes. (PDF 40 kb)Additional file 6Figure S6. Model-based clustering of HSMM dataset by any three highly variable genes. (PDF 68 kb)


In the last couple of years, schizophrenia was often discussed as autoimmune disease. Several antibodies were suspected, but so far there has been no proof of Gamma-aminobutyric acid (GABA) receptor antibodies in patients with schizophrenia.In this case report we present a 21-year old woman with schizophrenic symptoms, who showed anti-GABAB1 antibodies when screened by a vast recombinant neurology mosaic on Human Embryonic Kidney Cells 293 (HEK293) cells. The young woman presented with various psychotic symptoms as well as speech and motor ataxia, with the neurological signs starting in childhood.A hypofunction of the GABAergic system is a possible cause of severe schizophrenic symptoms. Postmortem studies proved this hypothesis by showing dysfunctional GABAergic interneurons in various brain areas. Therefore one should always think of an immune-mediated pathogenesis as well memory impairment and behavioral changes co-occur with frequent seizures. Schizophrenia is a severe polygenic and heterogeneous psychiatric disease with onset in early adulthood. According to the Diagnostic and Statistical Manual of Mental Disorders schizophrenia is characterized by profound disruption in cognition and emotion, psychotic manifestations and negative symptoms. The causes of schizophrenia have been the subject of much debate and the underlying pathomechanism remains so far unknown . IntrinsB is a G-protein coupled transmembrane receptor consisting of two subunits . Subunit B1 is responsible for the transmitter binding as well as for the general receptor function, whereas B2 ensures the transmembrane localization and the G-protein activation. A direct antagonistic binding to the GABA receptors (GABAR) and dysfunctional inhibitory cascade could imitate the same schizophrenic symptoms as a decreased level of the transmitter GABA itself.Glutamate is a key player in the mediation of schizophrenic symptoms and by non-competitively antagonizing the glutamate N-methyl-D-aspartate (NMDA) receptor with substances such as ketamine, phencyclidine, or Dizocilpine (MK-801) in animals , neuroloThe role of GABAR has already been shown in several immunohistochemical experiments in which reduced immunolabeling of GABAR subunits occurred in different brain regions  of schizophrenic patients , 7.B receptor antibody mediated encephalitis patients usually present with a classical limbic encephalopathy with seizures. Magnetic Resonance Imaging (MRI) findings typically show an increased signal in the medial temporal lobes, and Cerebrospinal Fluid (CSF) shows an elevated lymphocyte cell count. The animal model of GABAB receptor antagonism resulted in similar symptoms such as seizures, memory deficits, anxiety, and affective dysregulation in rodents [B receptor encephalitis also develops without cancer association and resembles other central synaptic autoimmunities. The presence of CSF antibodies in patients with both neurological and psychiatric deficits has been examined [B receptor antibodies were frequently found in previously seronegative patients with encephalitis and small cell lung cancer, they did not occur in patients with isolated cerebellar ataxia [In case of a GABA rodents . Lancast rodents . Howeverexamined , 11. Wher ataxia .The 21 year old woman was admitted to our psychiatric ward as an emergency for the first time in 2010, reporting hallucinatory voices as well as persistent visual hallucinations  and tried to harm herself by repeatedly smashing her head against the wall. Moreover, she demonstrated psychomotor agitation and suffered from alcohol intoxication with 1.98 promille. The psychotic symptoms remained after detoxication and the patient reported to have suffered from negative symptoms such as apathy and depressed mood during the previous six months. The patient was diagnosed with paranoid schizophrenia according to the international classification of disease (ICD-10), which is commonly used in clinical routine as well as psychiatric research. During the following three months the patient was admitted two more times, always in a floridly psychotic state with self-inflicted injuries and intoxicated with alcohol. Antipsychotic treatment comprised ziprasidone and quetiapine with no satisfactory effect. The patient was treated with ziprasidone only for a couple of weeks and in a low dose. Continuous was with quetiapine for almost two years in an initial dose of 500 mg and a maintenance dose of 100 mg daily, both without considerable effect. Dosage was reduced due to the patient’s pregnancy.The patients’ first psychiatric contact occurred 2004 at the age of 14, when she was brought to a child psychiatrist because of unstable interpersonal relationships, self-consciousness, identity, and behavior difficulties. Auto-aggressive tendencies started at the age of 12. Because of acute distress, difficult adjustment to social situations and somatic manifestations, three more hospital admissions to child and adolescent psychiatry became necessary subsequently, all lasting but a few days.According to her mother the patient was delayed in speech and motor skills and attended a school for learning-disabled children. At the time of consultation, a psychomotor retardation was still evident. The patient complained of frequent headaches. The findings of the neurological exam indicated an ataxia in speech (dysarthria) as well as motor coordination. In pediatric physical assessment somatosensory evoked potentials (SEP) recording  and electromyography (EMG)  were normal, electroencephalography (EEG) was normal according to age. Brain MRI and angiography did not show any space-occupying lesion or vascular malformation. There were no detectable alterations of the purine and pyrimidine metabolism and electrophoresis of lipoproteins did not reveal any pathological results either.One sample of serum was collected and serum was isolated by centrifugation (2500 x g/ 10 min) and kept at −80 °C until completion of recruiting phase. Serum was subsequently sent for diagnostic workup to the authors’ lab , and tested for the presence of antineuronal antibodies by means of a commercial immunofluorescence assay .B surface antigen in the assay -Tween and incubated in PBS-Tween for at least 5 min. Bound antibodies were labelled using Fluorescein-conjugated goat anti-human immunglobulines IgG, IgA or IgM antibodies for 30 min and washed as described before. Samples were classified as positive or negative based on the intensity of surface immunofluorescence of transfected cells in direct comparison with non-transfected cells and control samples. Central nervous system (CNS) tissues were assessed for the presence of characteristic staining patterns indicative of the presence of antibodies against neuropil or other neuronal antigens in the patient’s sample. In a dilution of 1:320 the patient’s serum still showed antibody binding to GABAThe patient refused plasmapheresis to remove the antibodies. Further diagnostic including CSF samples (to confirm the presence of receptor antibodies) and chest x-ray ) were not performed for ethical reasons, since the patient accidentally got pregnant.The Ruhr-University Bochum ethics committee approved the analysis (3250–08), and written informed consent was obtained from the patient.Several auto-antibodies against neurotransmitters and their receptors  have been found in serum or CSF of schizophrenic patients, thus suggesting an autoimmune etiological connection . Anti-doSo far there has been no proof of GABA receptor antibodies in patients with schizophrenic symptoms. In this case report we presented a young woman with schizophrenic symptoms, who was screened by a vast recombinant neurology mosaic on HEK293 cells and showed anti-GABAB1 antibodies. The young woman presented with various psychotic symptoms as well as speech and motor ataxia.The clinical coexistence of neurological and psychiatric features in patients with encephalitis has been known for quite some time, but recently cell surface antibodies have been found in patients with so far purely psychiatric symptoms. For example, specific receptor antibodies (anti- Voltage-gated potassium channel complex (VGKC), anti-N-methyl-D-aspartate receptor (NMDAR), anti-a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)) were found in (young) patients with first episodes of psychotic symptoms , 18. In The current case is only a single description of an association in a patient that was not neurologically treated. There can therefore be no conclusion about the general relevance of these antibodies in schizophrenia, but the need for screening further patients is evident.


Infective endocarditis is a heterogeneous condition whose incidence is rising. Despite advances in surgery and diagnostic methods, one-year mortality has not changed and it remains at 30%. Patients with prosthetic valve and intra-cardiac device–related endocarditis are being seen more frequently and this condition is difficult to diagnose with conventional microbiological and imaging techniques. The modified Duke criteria lack sensitivity in this group and should be supplemented with newer imaging techniques, including 18F-fluorodeoxyglucose positron emission tomography (18F-FDG-PET) and single-photon emission computed tomography (SPECT). In this article, we discuss these techniques and their role in the diagnosis of infective endocarditis. Of equal importance,staphylococcus now supersedesstreptococcus as the most frequent causative pathogen and the average age of affected subjects is rising5. Therefore, patients are more frail and co-morbid and frequently present with more aggressive forms of the disease. These trends seem likely to offset the benefits associated with earlier diagnosis and more aggressive surgical management. As a result, IE has a one-year mortality exceeding 30%, which has remained unchanged over the past 20 years and is higher than that of many common cancers7.Although infective endocarditis (IE) remains a rare condition worldwide, incidence is risingndition  and incr9. Although these criteria remain useful as a diagnostic aid, they are less sensitive in patients with prosthetic valve endocarditis (PVE), ICED infection10 and IE affecting the right side of the heart. Both major criteria are less sensitive in PVE and ICED: negative blood cultures are common (>20%) and transthoracic echocardiography (TTE) can miss pacemaker lead involvement and PVE in up to 60% of cases13. It is also important to note that the Duke criteria were originally designed as a research tool and should not supplant clinical assessment.Diagnosis of IE remains challenging, requiring a high index of clinical suspicion and appropriate investigation of high-risk patients. Both the European Society of Cardiology (ESC) and the American Heart Association recommend use of the modified Duke criteria  for the Regardless of the chosen diagnostic guideline, it is likely that a greater proportion of patients with PVE and ICED infection will fall under the ‘possible’ category   is the gold-standard second-line imaging modality for IE and features prominently in both European and American guidelines. It has a sensitivity of more than 90% for native valve and more than 85% for prosthetic and device-related endocarditis16. The technique has particular value in the detection of paravalvular abscess, valve dehiscence, regurgitation and perforation9 and is more specific (but not more sensitive) for the exclusion of IE (up to 100%) when compared with TOE17. 3D-TOE may help to identify patients at increased risk of embolism by providing a more accurate assessment of vegetation size18. Finally, by providing an ‘en-face’ valve view, 3D-TOE may facilitate more accurate surgical planning. Currently, studies supporting the use of 3D-TOE in the diagnosis and management of IE are limited by their small number and size and there have been no studies specifically focused on the use of 3D-TOE in PVE or ICED endocarditis. It is also important to note that the low frame rate of 3D-TOE may impair the detection of small vegetations19. Therefore, the technique should continue to be used as a supplement to conventional TTE/2D-TOE in most patients.Three-dimensional transoesophageal echocardiography (3D-TOE) is carried out with a multi-plane probe incorporating a 3D matrix array. It generates full-volume datasets that can be manipulated to visualise vegetations and defects in planes and at angles that are not possible with conventional TOE21. MSCT uses ECG gating to eliminate movement artefact during the cardiac cycle and reduce radiation exposure to 2–3 mSv per scan20. It is equivalent (and may be superior) to TOE for the delineation of paravalvular anatomy and detection of fistulae and abscess22 and is superior to TOE for the detection of mycotic aneurysms. Use of MSCT may also reduce the need for invasive imaging by characterising the coronary arteries, aortic valve, root and ascending aorta23. It is also less susceptible to prosthetic valve artefact than TOE and therefore is an attractive adjunct in the assessment of suspected PVE. In a group of 28 patients with suspected PVE, the addition of MSCT to routine work-up (including TTE/TOE) resulted in a major diagnostic change in six patients (21%)23. Unlike echocardiographic techniques, MSCT has the potential to consolidate the diagnosis of IE by identifying distal embolic complications if the scan window is extended beyond the thorax. Drawbacks include the need for exposure to ionising radiation, use of iodinated contrast and the lack of large studies comparing the technique with conventional echocardiography.Multislice computed tomography (MSCT) is complementary to conventional echocardiography in the diagnosis of IE and indications for its use have been incorporated into the 2014 American College of Cardiology/American Heart Association valvular heart disease guidelines  and the 2015 ESC guidelines on the management of IE24 (sensitivity of 87% and specificity of 92%)25. Supplementing the diagnostic sensitivity of the modified Duke criteria in suspected PVE and ICED endocarditis increased from 52 to 91% in association with the use of 18F-FDG-PET25, leading to its incorporation into the diagnostic algorithm for high-risk patients with equivocal initial investigations in the 2015 ESC guidelines9. Like MSCT, 18F-FDG-PET can detect distant emboli and evidence of metastatic infection, increasing sensitivity relative to TTE and TOE and identify higher-risk patients—those with more metastatic infection—who may benefit from early surgery26. 18F-FDG-PET has the advantage over MSCT in that it is normal practice to image the whole skeleton rather than just the thoracic cavity. However, as with all imaging techniques, 18F-FDG-PET results must be interpreted with caution. At present, it is not possible to distinguish a sterile, post-operative inflammatory response from infection. Nor can 18F-FDG-PET differentiate thrombi, soft atherosclerotic plaque or foreign body reactions. Given the high mortality associated with device and valve explantation, 18F-FDG-PET data should always be interpreted with caution, especially in the early post-operative phase21. Moreover, sensitivity is low in native valve IE and routine use is not recommended in this group27.18F-fluorodeoxyglucose positron emission tomography (18F-FDG PET) is a relatively new technique more frequently used in the diagnosis and staging of cancer that shows promise for improved assessment of ‘possible’ IE according to the modified Duke criteria. The technique highlights areas of increased glucose metabolism corresponding to active inflammation, which can be mapped to conventional computed tomography (CT) datasets to provide anatomical landmarks. Although early studies generated concerns with regard to low sensitivity in native valve IE (<40%), the technique has demonstrated significant value in the diagnosis of PVE and ICED endocarditis21. There have been relatively few large studies evaluating the utility of SPECT in the diagnosis of IE, but multiple case reports and smaller trials suggest benefit in PVE30. As a result, its use has been incorporated into the 2015 ESC guidelines as an adjunct to repeat TOE/TTE in possible PVE9. SPECT was more specific (100% versus 71%) but less sensitive (64% versus 93%) than 18F-FDG-PET CT in a cohort of 39 patients with suspected PVE30. However, increased specificity in patients who have undergone recent surgery offers advantages: in a group of 131 patients evaluated with SPECT for suspected IE (including those operated within two months), sensitivity was 90% and specificity was 100%28. Conversely, SPECT is more time-consuming than 18F-FDG-PET CT, requires blood handling for preparation of radiopharmaceuticals, and has lower spatial resolution than 18F-FDG-PET CT29. Sadly, current data are mostly limited to native and prosthetic valve endocarditis, so additional work will need to be carried out to establish the full role of SPECT in suspected ICED endocarditis.Single-photon emission computed tomography (SPECT) is a nuclear technique that measures gamma rays emitted from injected radionucleotides. Multiple 2D plane images are acquired and reconstructed to provide anatomical localisation in 3D. Detection of infection relies on the accumulation of neutrophils in the region of IE and the technique is most sensitive in the acute phaseThe landscape of IE is changing: older and increasingly co-morbid patients with prosthetic material are presenting to hospital with staphylococcal IE that can be difficult to detect by using conventional microbiological and imaging techniques. The modified Duke criteria  are neitMSCT and 3D-TOE are broadly complementary to conventional diagnostic work-up and may facilitate better surgical planning. In contrast, 18F-FDG-PET CT and SPECT have the potential to determine a diagnosis of IE, even in patients with prosthetic valves and intra-cardiac devices. SPECT is clearly more suitable for patients who have recently undergone surgery as it remains specific, whereas the higher sensitivity and simpler technical aspects of 18F-FDG-PET CT make it an attractive second-line test in patients with a high clinical likelihood of IE and normal or equivocal initial imaging findings. The increasing role and importance of nuclear medicine in the diagnosis and management of IE provide further support for a hub-and-spoke model of service provision, and complex cases are best managed in tertiary reference centres that have the experience and technical resources to offer these techniques routinely.