It has been known for some time that neutrophils are present in the tumour microenvironment, but only recently have their roles been explored.Comprehensive literature search of neutrophils and cancer  for key articles . References from these articles cross-checked for additional relevant studies.Neutrophils are a heterogeneous population with both pro- and antitumour roles, and display plasticity. Several neutrophil subpopulations have been identified, defined by a combination of features .Limitations in translating murine tumour models to human pathology and paucity of human data. Consensus in defining human neutrophil subpopulations.Neutrophils as therapeutic targets and as possible playmakers in the biological response to newer targeted cancer drugs.Understanding the metabolic programming of neutrophils in the tumour microenvironment. Tumours produce granulocyte colony-stimulating factor (G-CSF) which skews the neutrophil retention/release balance in bone marrow, leading to this increase in blood neutrophils.et al. comprising over 40 000 patients, showed NLR > 4 to be associated with worse overall survival, cancer-specific survival, progression-free survival and disease-free survival.5 This was seen in all types and stage of cancer.Many research groups have investigated whether the number of neutrophils present in peripheral blood correlates with patient outcome. Most have done this using neutrophil-to-lymphocyte ratio (NLR). A meta-analysis of 100 such studies by Templeton 6 In addition, Gentles et al. used a computational approach to analyse bulk tumour transcriptomes in order to infer the frequency of different immune cell populations (including neutrophils) in over 3000 solid tumours (14 cancer types). They found intra-tumoural neutrophils to be the most adverse prognostic cell population.7The association between peripheral blood neutrophils and survival does not however give any information about what might be happening at the tumour site itself i.e. whether tumour-associated neutrophils (TANs) are associated with outcome. A meta-analysis of nearly 4000 patients has shown high levels of intra-tumoural neutrophils to be associated with unfavourable survival.8 This may of course just be a reflection of adequate toxicity of the drug being achieved to kill tumour cells, however the question does arise as to whether the direct effect on neutrophils themselves is beneficial. This is clinically relevant, as the use G-CSF in patients who are profoundly neutropaenic post-chemotherapy (used with the aim to prevent sepsis) could in fact be detrimental to longer term clinical outcome. Indeed, it has been shown that G-CSF may promote metastatic disease via neutrophils forming a pre-metastatic niche.9In direct contrast, neutropaenia in patients undergoing chemotherapy has been shown  to be beneficial to survival.Measuring the number of neutrophils in the blood and/or in the tumour of cancer patients and associating this with survival is of course quite a crude measure and does not give any indication regarding mechanism. It must also be remembered that blood neutrophil levels increase under other conditions, such as infection. Within the same patient, neutrophils may display varying roles at different sites. Furthermore, appropriate inflammatory responses are dependent upon a functioning balance of neutrophil production, release from bone marrow, recruitment to the site of injury and clearance. Dysregulation of this homeostatic process, for example, by tumour-derived G-CSF, could perpetuate malignancy.10 Using IFN-β1−/− tumour-bearing mice, the Jablonska group have shown interferon beta (IFN-β) to have the opposite effect to TGF-β on TAN polarization, i.e. IFN-β promotes antitumour N1 TAN.11Fridlender was the first to suggest that TAN may be polarized to N1 (antitumour) or N2 (pro-tumour) phenotypes, in a similar manner to macrophages. Using tumour-bearing mice, he demonstrated that transforming growth factor beta (TGF-β) blockade favoured the accumulation of N1 TAN that were morphologically and functionally different to N2 TAN. N1 TAN had hypersegmented nuclei in contrast to the nuclei of N2 TAN that were circular. N1 TANs were cytotoxic to tumour cells via an oxygen radical-dependent mechanism and had increased tumour necrosis factor alpha (TNF-α) and intercellular adhesion molecule 1 (ICAM-1) expression, whereas N2 TAN expressed high levels of arginase which is known to suppress T cell immunity. Of key importance, TGF-β blockade promoted a T cell antitumour response.12 It seems increasingly likely that N1 and N2 represent laboratory extremes of a biological continuum, with plasticity dependent upon the local environment. Therefore rather than focusing on N1/N2, we should possibly instead be defining neutrophils by the distinct functional phenotypes/subpopulations that aid or abate the process of tumourigenesis  in the different microenvironments .It should be noted that the work described above in murine models is yet to be replicated in human TAN. Furthermore, in a similar manner to M1 vs M2 macrophage classification in malignancy, it may be that a binary N1/N2 classification of neutrophils is an oversimplification.et al. showed that NE is taken up by tumour cells, where it degrades insulin receptor substrate-1 (IRS-1). Lower levels of IRS-1 were associated with an increase in the interaction between phosphatidylinositol 3-kinase (PI3K) and the potent mitogen platelet-derived growth factor receptor (PDGFR), which directed the PI3K axis to favour tumour proliferation.13Neutrophil elastase (NE) has been shown to promote tumour proliferation. Houghton 14 Of note, the MET proto-oncogene is expressed in neutrophils and is required for neutrophil chemoattraction and cytotoxicity towards tumour cells in response to its ligand hepatocyte growth factor.15 Neutrophils can kill tumour cells via TNF-α expression.11 Furthermore, neutrophils stimulated by interferons release tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) which induces tumour cell apoptosis.16In contrast, neutrophils can also induce lysis of tumour cells via hypochlorus acid produced from reactive oxygen species (ROS).18 It is thought that neutrophils may drive angiogenesis in malignancy by providing a significant source of MMP-9 which acts to release VEGF from the extracellular matrix (ECM).20 In addition to roles in angiogenesis, MMP-9 is also postulated to aid the direct invasion of tumour cells via degradation of ECM/basement membrane.The angiogenic and invasive mechanisms of matrix metallopeptidase 9 (MMP-9), vascular endothelial growth factor (VEGF) and Bv8 (prokineticin) have been previously described.21Of note, it has been shown that neutrophil extracellular traps (NETs), formed during neutrophil death, and composing of chromatin, NE and myeloperoxidase, have a role in angiogenesis, by stimulating vascular endothelial cells to release proangiogenic cytokines.ex vivo to release the antiangiogenic isoform of VEGF (VEGF-A165b),22 but it is yet to be proven if this occurs in vivo.Countering the above mechanisms, neutrophils can also have an opposing function with regard to angiogenesis; it has been reported that neutrophils can be conditioned 23 Neutrophils have been widely reported to suppress T cell proliferation in ex vivo studies, with Coffelt et al. demonstrating this is inducible nitric oxide synthase (iNOS)-dependent,24 but it must be noted that recently concerns have arisen about the accuracy of these assays when T cell activating microbeads have been used.25 Neutrophils have also been shown to induce the apoptosis of CD8 T cells in a TNF-α- and nitric oxide (NO)-dependent manner.26 In addition, neutrophils can suppress T cells via programmed death ligand 1 (PD-L1).27 Moreover, neutrophil depletion studies suggest that they may act to reduce the effectiveness of PD1 immunotherapy.28 Finally, neutrophils recruit regulatory T cells into tumours via secretion of chemokine ligand 17 (CCL17), which may inhibit antitumour immunity.29As previously mentioned, neutrophils are known to express arginase. Arginase degrades arginine, an essential amino acid important in many cellular processes, e.g. the proliferation of T cells. High arginase levels can be found in the tumour microenvironment and result in inhibition of T cell receptor expression and antigen-specific responses, aiding tumour evasion.30 can stimulate T cell proliferation31 and suppress pro-tumoural IL-17 γδ T cells via ROS.32Whilst there appears to be an ever-expanding list of ways in which neutrophils immunosuppress in the tumour microenvironment, nonetheless neutrophils can also be immunostimulatory and antitumour. Neutrophils can have a role in antigen presentation,33 Extravasation into tissues is aided by interactions between β2 integrin on neutrophils and ICAM-1 on tumour cells, promoting anchoring to the vascular endothelium.34 Tumour-derived G-CSF can initiate a pre-metastatic environment in distant organs, mobilizing neutrophils from the bone marrow to swarm at the metastatic site before tumour cells arrive.35NETs have been shown to sequester circulating tumour cells at distant sites and promote metastasis.et al. showed that tumour-entrained neutrophils (i.e. stimulated by the primary tumour) can inhibit metastatic seeding via hydrogen peroxide (H2O2) killing of disseminated tumour cells.36 It has recently been shown that H2O2 from neutrophils kills tumour cells by triggering a lethal influx of calcium via transient receptor potential cation channel subfamily M member 2 (TRPM2), an H2O2-dependent calcium-permeable channel expressed on cancer cells.37Conversely, Granot 40It is clear from earlier sections in this review that neutrophils display heterogeneity and plasticity of function in malignancy. But what other features define these neutrophils that are pro- or antitumour? Eruslanov and Fridlender groups have carried out seminal work in the last few years where functional neutrophil subpopulations have been distinguished further by density, maturity, surface markers, morphology and anatomical site Fig. .30,31,38Ex vivo, neutrophils are commonly isolated from the blood by discontinuous density gradient. This results in the formation of a red cell pellet, a normal-density (sometimes referred to as ‘high density’ in the literature) layer of cells and a low-density layer of cells. Neutrophils are usually the predominant cell type in the normal-density layer  and peripheral blood mononuclear cells (PBMCs) are found in the low-density layer.41 In malignancy, there is an expanded population of neutrophils in the low-density layer , which can be further defined by maturity/morphology. LDNs are large and are either immature with a banded/ring nuclei, or mature with segmented nuclei. All LDNs are pro-tumour, displaying immunosuppressive properties. NDNs are small and mature with segmented nuclei and are antitumour. Of note, neutrophils from patients with malignancy resist apoptosis compared with healthy donor neutrophils. Finally, murine models showed LDN to originate from the bone morrow, but of interest TGF-β could also mediate the transition of HDN to LDN (displaying plasticity).4038The features of TAN in murine models have already been described. In humans, TANs in early-stage cancer are mature with segmented nuclei and are antitumour, activating T cell responses. Study of TAN from more advanced malignancies is proving more difficult, due to lack of availability of tissue to study .42 However, most groups use CD11b+CD15+CD66b+CD14− to identify neutrophils in humans. In addition, CD10 is proving to be a key marker for the maturation and suppressive potential of neutrophils. Indeed, going forward CD10 may reduce the need to use density gradients to define neutrophil populations.43To date, it has proven difficult to distinguish between the neutrophil subpopulations described above, by surface marker immunotyping. Furthermore, there have been difficulties when attempting to compare neutrophil subpopulations found in murine models with those found in humans.+CD14-CD15+(or CD66b+)CD33+, it has been difficult to distinguish between them. However, it is known that G-MDSCs are found in the low-density fraction of peripheral blood. Furthermore, G-MDSCs express lectin-type oxidized LDL receptor 1 (LOX-1) and so this marker may be used to distinguish them from neutrophils, without the need for a density gradient.45The name myeloid-derived suppressor cells (MDSCs) was originally coined over 10 years ago to describe a group of myeloid cells with immunoregulatory activity, i.e. suppressed antitumour T cell functions (via arginase and ROS). Broadly speaking, they compose two phenotypical/morphological groups of cells: those similar to neutrophils (granulocytic/polymorphonuclear MDSCs or G-MDSCs/PMN-MDSCs) and those similar to monocytes (M-MDSCs). It is thought that during chronic inflammatory processes, such as malignancy, there is a persistent signal to recruit neutrophils and monocytes from the bone marrow . As time goes on, the rate of demand on the bone marrow is such that these recruited cells are increasingly immature and have aberrant function. These cells are MDSC, and as cancers progress they form a greater proportion of circulating cells. In humans as G-MDSCs and neutrophils can both be defined by the surface markers CD11b46 It seems increasingly likely that in humans, immature peripheral blood LDN and G-MDSCs are one and the same. However, mature peripheral blood LDN appear to be different to G-MDSCs. Furthermore, within tumours themselves the relationship between G-MDSCs and TAN is entirely unknown.47There is debate as to how closely neutrophils and G-MDSCs are related.Neutrophils are no longer seen as a simple first responder cell; they have complex multifaceted roles in all stages of malignancy with both pro- and antitumour roles. In cancer, neutrophils are a heterogeneous population and display plasticity. Several neutrophil subpopulations have been identified and are currently defined by a combination of features; density, maturity, surface markers, morphology and anatomical site..12Our summary figure Fig.  aims to 42 These mainly relate to (i) the limitations of translating murine tumour models to human pathology and (ii) the lack/quality of human data.A recent review article by the Eruslanov group highlights the ongoing areas of controversy regarding the role of neutrophils in cancer.The most common form of murine model is transplantable tumours, i.e. immortal cell lines that have undergone years of selective pressure and are chosen based on their ability to grow quickly when injected into the mouse. These tumours by definition have a very different natural history to the gradual evolution of a natural tumour and therefore really only represent the later stages of malignancy. There are no shared neutrophil cellular markers between mouse and man. We do not know if immature LDN, G-MDSCs and N2 are the same cell population or not. N1/N2 polarization has not been shown in human TAN, and it seems more likely that in fact there will be a continuum of behaviour rather than these two extremes. Are N2 neutrophils just immature cells recruited as a consequence of cancer and not a contributing cause?ex vivo T cell responses which may not reflect true physiology. For example, as previously described there are concerns about artefact that may be created by the methodologies used for ex vivo T cell proliferation studies.There is a lack of human tissue available from the later stages of cancer, as patients do not routinely have surgery. Furthermore, whilst peripheral blood may be more easily available from these patients (than solid tumour samples), the behaviour of blood neutrophils may be completely different to that of TANs. When tumour samples are obtained, it must be noted that neutrophil function can be changed by the process of disaggregation used to extract the neutrophils. Finally, in human studies, a lot has been attributed to Overall, there needs to be an unpicking of the various neutrophil subpopulations, and a better understanding of their evolving roles as cancers progress, at both primary and metastatic sites.48 photodynamic cancer therapy49 and Bacillus Calmette–Guerin immunotherapy.50 However, a growing area of research is considering neutrophils as a therapeutic target themselves. Whilst the short-lived nature of neutrophils and their essential role in host defence against infection will need to be considered, nevertheless, targeted therapies relating to neutrophil recruitment, function and polarization (targeting the pathways mentioned in this review) may be an attractive add-on therapy to conventional treatments (i.e. chemotherapy/radiotherapy) and newer immunotherapies. For example, in terms of neutrophil recruitment, there is an interest in targeting the CXCR2 pathway.51 Relating to neutrophil polarization, there have been trials of TGF-β inhibitors as a cancer therapy.52 With regard to direct neutrophil–tumour interaction, there has been interest in the use of NE inhibitors.53In terms of established therapies, neutrophils have been shown to be important players in the beneficial immune response to antibody-based cancer therapy,15 but others hopeful of benefit.54 As previously mentioned, neutrophils have been shown to express PD-L1, and therefore immune checkpoint inhibitors may have a role in reducing neutrophil suppression of T cell responses.Finally, an expanding area of interest is understanding how neutrophils respond to newer targeted cancer therapies. Inhibitors of the receptor tyrosine kinase c-MET have been shown to inhibit neutrophil recruitment to tumours, with some concerned this may be detrimental,55 It is known that the tumour microenvironment has altered oxygen and metabolite availability. Oxygen-sensing pathways and metabolic flux regulate neutrophil function and survival responses.57 There is literature regarding metabolic reprogramming of tumour-associated macrophages.58 Of note, MDSCs, which have infiltrated tumours, increase fatty acid uptake, and inhibition of fatty acid oxidation blocks their immunosuppressive function.44 Whilst there is extensive literature regarding the role of MDSCs in hypoxia, whether metabolite availability or hypoxia in the tumour microenvironment plays a role in the functional polarization of neutrophils is yet to be explored.It has been shown that during tumour progression, TAN distribution is more within the tumour, and TANs develop pro-tumourigenic properties.


Several publication databases now index the associated funding agency and grant number metadata with their publication records. Librarians who are familiar with the particulars of these databases can assist investigators and administrators with data gathering for publication summaries and metrics required for renewals of and progress reports for National Institutes of Health (NIH) grants.Publication lists were pulled from three main indexers of publication-associated funding information , using iterative search strategies. All discovered variations for the cited grant number of interest were recorded and tested. Publication lists were compared for overall coverage.A total of 986 publications citing the single grant number of interest were returned from the given time frame: 920 were found in PubMed, 860 in NIH RePORTER, and 787 in Web of Science. Web of Science offered the highest percentage of publications that were not found in the other 2 sources (n=63). Analysis of publication funding acknowledgments uncovered 21 variations of the specific NIH award of interest that were used to report funding support.This study shows that while PubMed returns the most robust list of publications, variations in the format of reported funding support and indexing practices meant no one resource was sufficient to capture all publications that cited a given NIH project grant number. Librarians looking to help build grant-specific publication lists will need to use multiple resources and be aware of the most frequently reported grant variations to identify a comprehensive list of supported publications. Funding awards from the US National Institutes of Health (NIH) typically require publications and other outcomes of the grant to be regularly reported to remain in good standing throughout the award period. Similarly, for renewal considerations, a comprehensive list of publications produced under the award are often needed to demonstrate successful outcomes and the impact of funded research. To support these requirements, large multidisciplinary research centers may turn to their affiliated libraries for assistance in identifying publications produced under their NIH program projects or center grants. Such program grants  are large, multi-project efforts that may support several hundred researchers with various community and institutional affiliations. Searching in the public record remains an accepted and reliable way to identify publications that researchers produce under such funding awards. However, searchers need a clear understanding of how NIH awards of interest are indexed in various data sources to build effective search strategies.Since first offering funding acknowledgment indexing in 2008, Web of Science remains the most widely recognized and recommended source for analyzing publication funding acknowledgments –10. HoweNIH Research Portfolio Online Reporting Tool for Expenditures and Results (NIH RePORTER) provides an additional means of identifying supported publications.Comparisons of acknowledgment indexing between Web of Science and PubMed highlight the language, country of origin, document type, and disciplinary biases found in both databases , 7–9, 12This study describes an attempt to construct a comprehensive list of publications produced by research members under an NIH program award to the Center for AIDS Research (CFAR) at Emory University. This center reached out to the Woodruff Health Sciences Center Library for assistance with preparing the necessary publications list for an upcoming grant renewal. After initially pulling publications from NIH RePORTER, it was noticed that many grant-citing publications were overlooked. Additional publications were, therefore, sought from both Web of Science and PubMed. The publication lists provided by these three data sources were compared to improve understanding of current database indexing practices and help others build appropriate search strategies for identifying specific funding award–associated publications. The goals of this study were (1) to identify the level of coverage provided by each data source and (2) to design appropriate search methods that could be adapted for future inquires of NIH grant-supported publications.NIH RePORTER provides lists of publications produced as a result of NIH support. The NIH RePORTER public query interface was used to search for all results under grant number P30AI050409 since 2008, and all results under the “publications” tab were exported for review. As of the collection date of June 2018, a total of 860 publications were listed as being published between 2008 and 2017. PubMed identification numbers (PMIDs) for these publications were exported.Publication lists in NIH RePORTER are mapped to a single standardized identifier, or grant number, issued by NIH. By contrast, funding acknowledgments in Web of Science and PubMed are based in part on free-text indexing, which is subject to grant number variations. To illustrate, Initial identification of Web of Science search terms was performed using the NIH RePORTER dataset. A Web of Science results list was created using an advanced search query by the PMIDs provided by NIH RePORTER. The “Analyze Results” feature was then used to collect and record the relevant variations in funding acknowledgment. This feature is available under the “Analyze Results” link at the top of all Web of Science results lists: users can select the “Grant Numbers” filter to view and export all indexed grant numbers associated with the search results. All P30AI050409 variations were recorded. These recorded variations were then tested in a new search string to identify additional indexed publications that acknowledged the grant of interest but were not listed in NIH RePORTER. Variations discovered in PubMed were also tested and added as appropriate.Further variations, given observed patterns, were also tested . Wildcard (*) searching was attempted but cannot be recommended, as only right-hand truncation  is currently supported in the grant number search field and generally returned results unrelated to the award of interest. All required keywords for a comprehensive publication list were recorded, revealing 17 variations to the CFAR grant number in 787 publications. All publications returned by these keywords were found to correctly relate to the award of interest. The final Web of Science search strategy was:fg=(50409 OR 050409 OR AI50409 OR A150409 OR AI050409 OR A1050409 OR AI0050409 OR A01050409 OR P30AI50409 OR P30AI050409 OR P30A1050409 OR P30AL050409 OR 2P30AI050409 OR 2P30A1050409 OR 5P30AI50409 OR 5P30AI050409 OR 5P30A1050409)PubMed advanced search options provide a list of unique grant numbers. By entering leading characters into the “Grant Number” field and selecting “Show Index List,” users can browse possible complete number variations. From this index, variations to the P30AI050409 grant number were tested and added to a saved search string, and publication results were examined. Attempts were made to discover all listings including the numeric string “50409,” and results were reviewed for matches to the award of interest. This was done through manual exploration of the index list, as left-hand truncation, internal truncation, and wildcard searching (*) are not currently supported by PubMed. Right-hand truncation  returned results that were not related to the grant of interest or were more accurately captured through more specific keywords .Variations discovered in the Web of Science database were also tested and added as appropriate. Hypothetical variations, given observed patterns, were also tested . In total, 46 variations in the CFAR grant number were discovered, returning 920 publications from 2008 to 2017. Results returned by each variation were recorded, reviewed, and compared; this process revealed that 15 unique keywords were needed to return a comprehensive publication list. For example, while ai050409-04a1 (denoting the 4th year of active funding) and ai050409-07 (denoting the 7th year of active funding) were listed separately in the advanced search index, a search for “ai050409” returned both, meaning that these 2 variations required only a single keyword. All 920 publications returned by these keywords were found to correctly relate to the award of interest. The final PubMed search strategy was:((((((((((((((“50409 04a1”[Grant Number]) OR “50409 10”[Grant Number]) OR ai50409[Grant Number]) OR ai050409[Grant Number]) OR ai0050409[Grant Number]) OR aL050409[Grant Number]) OR a150409[Grant Number]) OR a1050409[Grant Number]) OR a01050409[Grant Number]) OR p30ai50409[Grant Number]) OR p30a1050409[Grant Number]) OR p30AL050409[Grant Number]) OR “2p30ai 050409”[Grant Number]) OR 2p30a1050409[Grant Number]) OR 5p30ai50409[Grant Number])A total of 986 unique publications acknowledging the award of interest were identified from PubMed, Web of Science, and NIH RePORTER. While PubMed returned the largest number of publications, Web of Science returned the most publications that were not identified by the other 2 data sources. Although this observation held true across all 10 years, Web of Science offered a noticeably larger proportion of unique results in more recent years . Of thesVariations in funding acknowledgments stemmed primarily from truncations of the official NIH grant number . The mosOf the 3 investigated data sources offering lists of publications supported by the CFAR award of interest, none were sufficient to identify all publications supported by the award. PubMed returned the greatest number of results , whereas WoS returned an additional 6% that were not listed elsewhere. The NIH RePORTER listings captured only 87% of identified publications that acknowledged support from the CFAR award. The presence of unique results in other databases, regardless of publication age, suggests that this discrepancy was likely due to different indexing practices, rather than different data source processing times. Each data source identifies and indexes funding acknowledgments using different methodologies: NIH RePORTER relies on several extant databases such as eRA databases, MEDLINE, PubMed Central (PMC), the NIH Intramural Database, and iEdison ; PubMed Perhaps most surprising was that the publications that NIH RePORTER overlooked appeared in MEDLINE and PMC, despite both data sources being described in NIH RePORTER documentation. Variations in grant number format, while an obvious source of difficulty, could not, therefore, fully account for the discrepancy between databases. Of the publications that NIH RePORTER captured, 95% listed the variation AI050409, yet 48% of those indexed in PubMed but overlooked by NIH RePORTER included this same variation. Even when the funding acknowledgment is correctly listed in the full text or correctly appears in the PubMed record, the acknowledgment might still be overlooked by other databases. Considering these observations, it is recommended that multiple data sources be used when attempting to construct a comprehensive bibliography of publications that result from funding support.Despite the indexing discrepancies of all three data sources, the biggest difficulty in identifying publications produced by a particular award is author-supplied variations in funding acknowledgments. Thus, the greatest challenge to searching multiple data sources is creating effective search queries for each source. The findings presented here highlight some of the most common truncations and errors that are made when acknowledging NIH funding support, including expanded or truncated grant numbers, missing or extra zeros, and alphanumeric substitutions.These observations could be applied to inform likely keywords for identifying publications that acknowledge other NIH awards of interest. However, given the unpredictability of human error and in the absence of standardization in indexing acknowledgment fields, initial manual testing for likely variations of each specific grant number is recommended. At the very least, searchers will need to execute an iterative process of searching, identifying common errors, and expanding on likely substitutions to ensure the most complete list of publications. Furthermore, when searching by other grant numbers, particularly those using non-NIH structures, these recommendations may need to be modified to account for false positives .Finally, it is important to recognize that the formal reporting of funding support is still subject to disciplinary differences, organizational cultures, and individual author diligence. Thus, even the most robust search strategies might not identify all publication outputs from a given award. Studies as recent as 2015 report that only about 50% of natural sciences publications include formal support acknowledgments . Searchi


Microfabrication technology for cortical interfaces has advanced rapidly over the past few decades for electrophysiological studies and neuroprosthetic devices offering the precise recording and stimulation of neural activity in the cortex. While various cortical microelectrode arrays have been extensively and successfully demonstrated in animal and clinical studies, there remains room for further improvement of the probe structure, materials, and fabrication technology, particularly for high-fidelity recording in chronic implantation. A variety of non-conventional probes featuring unique characteristics in their designs, materials and fabrication methods have been proposed to address the limitations of the conventional standard shank-type (“Utah-” or “Michigan-” type) devices. Such non-conventional probes include multi-sided arrays to avoid shielding and increase recording volumes, mesh- or thread-like arrays for minimized glial scarring and immune response, tube-type or cylindrical probes for three-dimensional (3D) recording and multi-modality, folded arrays for high conformability and 3D recording, self-softening or self-deployable probes for minimized tissue damage and extensions of the recording sites beyond gliosis, nanostructured probes to reduce the immune response, and cone-shaped electrodes for promoting tissue ingrowth and long-term recording stability. Herein, the recent progress with reference to the many different types of non-conventional arrays is reviewed while highlighting the challenges to be addressed and the microfabrication techniques necessary to implement such features. Microfabrication technologies for neural recording have rapidly advanced over the past few decades in terms of spatial resolution ,2,3,4,5,Among a variety of MEMS-based neural probes with diverse geometries, materials, and fabrication techniques, the “Utah” and “Michigan” probe types illustrated in While these conventional cortical microelectrode arrays have been extensively used in a number of animal and clinical studies ,123,124,Various research groups have addressed these issues by introducing unique features which are added to conventional shank-type cortical arrays through specially devised microfabrication techniques. As illustrated in While a number of review papers have thoroughly discussed the design, fabrication, and testing results of conventional microelectrode arrays for the cortex ,206,207,Conventional MEMS-based microelectrode arrays typically carry neural interfacing electrodes only on a single side of the probe shaft. This stems from the nature of MEMS technologies, where each conductive or insulating layer is sequentially deposited and patterned on top of a substrate material. Neural signals from the backside of the probe are partially shielded by the probe shank, limiting the volume of tissue being recorded or stimulated to the neurons facing the front of the array. Recently, many research groups have developed multisided cortical arrays in which two or more sides of array shanks are formed with microelectrode sites by introducing special schemes during the microfabrication process. These multi-sided neural probes can provide the advantages of an increased recording volume without a shielding effect as well as an increased number of sites without significantly increasing the probe size under the same line width or spacing.Dual-sided arrays with sites on both the tops and bottoms of the probes have been proposed by several groups ,138,139.2 layer as a release layer and a site place holder, as shown in A three-sided array was developed by Seymour et al. . ElectroVarious types of brain probes with tube- or cylindrical structures have been demonstrated, offering several advantages over conventional shank-type probes. The hollow core of the tube structure can be utilized to add multi-functionality by inserting optic fibers or flowing drugs through the channel without increasing the probe dimension ,149,150.Fabrication techniques of three-dimensional tubes or cylindrical structures can generally be categorized into three types: creating a conventional planar thin-film array and wrapping it around a shank ,144,150,While non-planar photolithography techniques for the direct patterning of microstructures onto the curved surfaces of polymer tubes require complicated tooling, this non-planar patterning method can create a tube with a smaller diameter (~300 μm) with high throughput and without a manual rolling process. Typical non-planar microfabrication setups involve a UV laser projection system with a synchronized multiaxial motion stage for uniform exposure throughout the entire surface in addition to a rotation mechanism for metal deposition and photoresist coating, as presented in A tube structure could also be created by a series of special MEMS processes on a planar substrate by deep silicon etching and parylene deposition to form sidewalls and a ceiling, followed by XeF2 silicon etching to create a hollow space, which could be used for drug delivery or optic fiber insertion .The functionality of these tubular probes haven been demonstrated in animal tests using rats ,147,149,Conventionally, three-dimensional recording probes with both lateral and depth-wise access in the cortex could be fabricated typically by the stacking of multiple two-dimensional silicon probes, which requires precise assembly and interconnection schemes ,83. ThesFlexible polymer-based foldable arrays have been developed to address this issue, in which a non-planar structure could be created by folding some parts of the thin-film array by means of magnetic force ,154, eleThin layers of micromachined magnetic materials, such as electroplated nickel  or evapoA completely different concept of folding was also proposed when researchers introduced an origami-like method to combine epicortical ECoG and an intracortical depth probe into a single device . The plaOne of the major challenges related to the chronic implantation of conventional probes is tissue damage and scar formation around the implanted probe due to the considerable mechanical mismatch between the tissue and the silicon probes, and the forces exerted by the micro-motions of brain tissue after implantation, all of which eventually cause degradation of the signal quality. Flexible thin-film polymeric probes can be used for high conformability to brain tissue, but they are not rigid enough to penetrate into the cortex such that a special delivery tool or stiffening by biodegradable materials is required. Mechanically dynamic probes have been demonstrated to overcome the abovementioned limitations, with some parts of the probes designed to self-soften or self-deploy in response to physiological conditions using thermally sensitive or water-sensitive composite materials ,159,160,Self-softening probes are initially stiff enough to facilitate cortical insertion with minimized trauma, but they become mechanically compliant post-implantation to closely match the modulus of the brain tissue while minimizing mechanical stress and glial reaction. This transition of the mechanical properties triggered by heat or water can be implemented using special composite materials as a probe substrate, including nanocomposites ,158,159,Some parts or the entire array of self-deployed probes can physically move toward target locations populated by neurons that are more viable and less impaired by the insertion of a probe shank ,167,168,Minimizing the reactive tissue response is the key factor during the chronic recording of neural activity ,145. TheHighly compliant sub-μm mesh electronics have been demonstrated to be capable of minimizing the stress exerted onto the tissue while also allowing neural cells, nutrients, and cytokines to interpenetrate through the open space of the mesh ,216. Acu2 hosting up to 16 recording sites along the shaft, as shown in The nanoelectronic thread (NET) electrode is an SU-8-based ultrathin thread-like single strand with a typical cross-sectional profile of ~ 0.5 × ~ 10 μmOther common approaches for reducing glial scarring involve the use of flexible polymer-based probes with minimized cross-sectional areas, temporarily strengthened by biodegradable supporting materials during insertion ,180,181.Cortical probes with microscale or nanoscale surface texturing have been proposed to reduce the immune response and prompt neural integration near recording sites for improved chronic performance ,183. NanMicron-scale surface structures such as microwells ,187 and Parylene C neural probes with a three-dimensional sheath structure were demonstrated to have a truncated cone at the tip of the probe with a length of 800 μm, a base diameter of 300–450 μm, and a tip diameter of 50 μm. The goal of this was the ingrowth of neural cells and thus enhanced tissue integration around the recording sites lining the interior and exterior regions of the cone sheath ,191. TheUndoubtedly, stable neural recording during the chronic implantation of intracortical microelectrode arrays is of paramount concern for the successful implementation of neural prosthetic devices such as brain-machine interfaces (BMIs) and for a better fundamental understanding of brain functions. Despite the fact that the field of microfabricated cortical probes represented by Utah or Michigan arrays have witnessed remarkable advancements over the past few decades, there remain technical challenges and limitations resulting in degraded or inconsistent recording quality levels and/or electrode failures over time. The approaches introduced in this article are in the form of a summarized overview of the various types of unconventional cortical probes featuring unique design characteristics which specifically overcome the limitations of conventional shank-type probes with regard to chronic cortical recording. These unconventional designs include multi-sided arrays, mesh- or thread-like arrays, tube-type or cylindrical probes, folded arrays, and self-deployable probes, with the common goal of stable and high-quality recording of neuronal activity from the brain for extended periods of time. The new ideas applied to these unconventional probes have been significantly effective to address various targeting issues by minimizing neural damage during and after insertion, adding the potential capability of multi-modality, and increasing the three-dimensional recording volume.It should be noted, however, that none of the abovementioned technologies can resolve all of the challenges at once. Therefore, major technical breakthroughs are expected in the fields of neural engineering and neuroscience by those who incorporate many existing solutions into a single device, enhancing our understanding of the brain and bringing the practical use of BMI technology far closer to reality. This is why we should be encouraged to continue to pursue new technical developments and exciting solutions to meet the requirements of a high-density and high-fidelity cortical interface for brain science and engineering.Although this review is focusing on the microfabricated probes, a variety of arrays not fabricated through MEMS technology also comprise an important part in the field of cortical probes. These types of probes are typically based on precise assembly of bundle of wires such as thin metal wires, carbon fibers, carbon nanotube (CNT), and/or optic fibers to create neural interfaces with high-density and multi-modality ,223,224.


Dietary trends and changing lifestyle patterns have been associated with the increasing occurrence of obesity in the Western world. These dietary trends are commonly studied using longitudinal food consumption surveys. An alternative to studying changes in eating behaviour may be found in recipe analysis of traditional cookbooks. Few such studies exist, however, and it is unclear whether dietary trends over time can be identified this way. The present paper analyses full-meal recipes from a traditional Dutch cookbook between 1950 and 2010. The selected recipes show an increase in energy density. Additionally, the protein weight per kcal increased. In general, the observed trends are similar to those identified by the Dutch National Food Consumption Survey. The analysis therefore suggests that traditional cookbooks can be used as an indicator to identify dietary trends over time, although further studies are necessary to support this hypothesis. Similarly, the study of Cohen et al. observes an increase in overall energy intake in the USA, with a percentage-wise decrease of energy from fats, mainly due to an increase in the amount of energy from carbohydrates. In Europe, the overall energy intake and the consumption of meat, fruit, vegetables, nuts and oils increased between 1961 and 2004, while changes in the intake of other food groups differed between Northern, Central and Mediterranean Europe. However, for the specific case of the Netherlands, Hulshof et al. observed a reduction in overall energy intake, while the percentage-wise intake of energy from plant proteins and carbohydrates increased between 1978 and 1997.The increasing occurrence of obesity in the Western world has led to studies analysing the changes in dietary patterns of the population over time. A recent report from the United States Department of Agriculture concludes that there was an increase in food intake from all food groups between 1970 and 2010 found an increase in mean energy content per serving size and an increased average energy density per serving between 1936 and 2006. Similarly, Eidner et al. observed an increased energy content per portion in Danish recipes between 1909 and 2009. Furthermore, an increase was observed within the food group intake of meat, starch products and sauce. The observed trends in both studies are in line with the conclusions on changing dietary patterns mentioned above. This raises the question whether recipes in a Dutch cookbook similarly reflect the observed changes in dietary patterns in the Dutch context.As an alternative to studying changing dietary patterns through nutritional surveys, cookbook recipes could provide an indication of the types and quantities of foods consumed at the time of publishing. However, only a few studies are available in the literature. The (retracted) analysis of a US cookbook by Wansink & PayneIn this brief report we compare recipes from a traditional Dutch cookbook over the period from 1950 to 2010 based on their nutritional content and we discuss the trends observed in this analysis in relation to those found by the Dutch National Food Consumption Survey (DNFCS).. The first edition of this cookbook was published around 1945 (exact date unknown) whereas the latest edition was published in 2010. For this study the edition from 1950 , and those from 1970, 1989 and 2010 were selected(–).The Dutch cookbook ‘Margriet Kookboek’ contains basic recipes for daily usage in a Dutch household. According to the publisher, more than 1·2 million copies were sold over the last decades, giving it a high coverage of the 7·86 million Dutch households counted by the Dutch Central Bureau of Statistics. Although most dinners will contain the three mentioned elements, there are exceptions such as a soup that functions as main course, or a cheese fondue. Recipes that did not describe a main course for dinner, or which according to their description would require additional dishes to form a full meal were excluded from the analysis.Recipes were selected based on their suitability to serve as a full meal according to the standards for a Dutch main course. A typical Dutch dinner consists of a starch source, vegetables and a protein product. In former times, most Dutch dinners contained potatoes, vegetables and meat (if it could be afforded), whereas currently more varied starch and protein sources are used . For items not covered by these lists, the Dutch Food Composition Database (DFCD) and the digital food monitoring application of the Netherlands Nutrition Centre were used. The nutritional content of the selected recipes was calculated based on the DFCD. The form to enter the recipes into a spreadsheet was coded using Visual Basic for Applications and Microsoft Office Excel. An example of the worksheet and the resulting database of recipes are available as Supplementary material.The ingredients of a recipe were classified into several groups , and a standardised form was used to express the more qualitative descriptions (e.g. one onion) in quantitative terms. For this, an internal cookbook weight reference list was usedP value of 0·05 for the significance level. When a significant difference was found, a post hoc analysis was performed with the Mann–Whitney test. For this test, the P value has to be adapted based on the number of pairwise comparisons executed. Comparisons with the Mann–Whitney test were performed for 1950–1970, 1970–1989, 1970–2010 and 1989–2010, with the adjusted P value of 0·0125 for the significance level. All considered recipes from the 1950 edition still existed unchanged in the 1970 edition, which is an edition extended with new recipes. Due to the high similarity of the recipes in the 1950 and the 1970 edition, the pairwise comparisons for 1950–1989 and 1950–2010 were omitted to reduce the number of required Mann–Whitney tests.Data were analysed using the software package SPSS statistics 23 (IBM). Levene statistics showed that data did not have homogeneity of variances. Therefore, comparison between all cookbooks was done with the Kruksal–Wallis test, with a In total, 187 recipes from the four different cookbooks met the selection criteria described in the previous section and were further analysed. The number of recipes selected per cookbook, the average energy content per portion and the portion weight are presented in Regarding meat, by far the most recipes contained pork (eighty-seven out of 187), followed by beef (forty-one out of 187), while thirty-eight recipes did not contain any meat or fish. Looking at animal-based ingredients in more detail, the recipes in the cookbooks of 1950 and 1970 contain ingredients such as lard, while these are not found any more in the recipes from the 1989 and 2010 cookbooks. Additionally, the later cookbooks contain more recipes using easy-to-cook meat, such as chicken. While none of the selected recipes from the 1950 cookbook contained poultry, 25 % of the recipes in the 2010 cookbook did. Later recipes also contained ingredients which were traditionally not used in Dutch cooking, such as olives.The observed overall decrease of portion weight per recipe can be explained by a decrease in the weight of staple foods and vegetables used. The decrease of these generally bulky ingredients also explains the observed reduction in fibre content. Additionally, the decrease of staple foods and vegetables leads to a relative increase of protein ingredients, which are more energy dense. This is also observed by the decreased portion weight per 100 kcal of food. Hence, the reduced portion weight does not lead to a similar reduction of the energy intake per meal.Given the similarities between the cookbooks of the years 1950 and 1970, the observed differences between the years 1970–1989 and 1970–2010 are expected to exist between 1950–1989 and 1950–2010 as well.et al., but contrary to studies from other countries which found an increase in energy intake. At the level of ingredient groups, our results are in line with the DNFCS. However, although a percentage-wise increase in the energy intake of protein was observed, the cookbook analysis suggests this is related to an increased contribution of animal protein, whereas Hulshof et al. observed an increase in plant-based protein. The increased contribution in the newer cookbooks of easy-to-cook animal protein corresponds to the shift in practice away from red meat. The total meat consumption has continued to decline slightly in recent years, which may be reflected by an increased number of non-meat or low-meat recipes in future editions of the cookbook.Our study shows a decrease in energy per recipe over the years, which is in line with the DNFCS report of Hulshof . Carbohydrates make up a large part of the energy consumption in-between meals, which nowadays accounts for about 30 % of the daily energy intake, equalling the carbohydrate consumption at dinner. Hence, the analysis of main meals was not able to detect this trend, showing a limitation of not taking into account the entire diet.A decrease was observed in the contribution of carbohydrates and fibres to the overall energy intake, where the DNFCS shows an increase in the contribution of carbohydrates. Trends in the consumption of these food items are therefore more likely to be spotted in the recipe analysis. Although the Dutch are suggested to be conservative in their eating behaviour, an increasing quantity of food seems to be consumed in-between the typical meals. However, this trend was not significant yet for most of the time period covered in this study.In the Dutch diet, food items such as potatoes, vegetables, legumes, meat, condiments and sauces are predominantly eaten during dinner. Hence, we conclude that this type of traditional cookbooks is likely to follow actual changes in dietary patterns. It may therefore be possible to use the historic developments in traditional recipes as an indicator of the actual changing dietary patterns over time. This is useful for those cases in which there are no detailed data available from food consumption surveys, although more study is required to further support this observation.Despite these limitations, the general trends observed in the cookbook analysis correspond to the trends identified in the DNFCS. Previous recipe analysis studies also observe a similarity between trends identified in recipes and trends identified in food consumption surveysNot all cookbooks will be suitable for such analyses, and the characteristics of suitable cookbooks require further investigation. Additionally, it is not clear how well recipes reflect home cooking practices, as consumers will often deviate from described recipes, or only use them for inspiration. Another limitation to the comparison of recipes throughout time is the possibility of variations in measures, and the interpretation of recipe instructions. The quantities of ingredients lost and wasted, the nutritional content, processing, etc. may differ throughout the years. These variations could affect the results of the analysis.et al., the relationship between recipes in cookbooks and home cooking practices can be investigated. Additional insights can be obtained from a further investigation of meal elements which combine into a meal, of combinations of meals for a daily consumption pattern, and of multi-day menu planning. This would aid the field of diet modelling, and could for instance be used by cookbook publishers to provide an accompanying web application which suggests recipes that improve an overall nutrient score  based on the meals selected by the consumer on the previous days.The results of this cookbook analysis raise the interest in studying other cookbooks and comparing them with results from dietary surveys. An extension of these analyses to the level of micronutrients would also be of interest. Moreover, as suggested by Eidner


Magnetic resonance imaging (MRI) allows to non-invasively evaluate rectal cancer staging and to assess the presence of “prognostic signs” such as the distance from the anorectal junction, the mesorectal fascia infiltration and the extramural vascular invasion. Moreover, MRI plays a crucial role in the assessment of treatment response after chemo-radiation therapy, especially considering the growing interest in the new conservative policy . We present a practical overview regarding the state of the art of the MRI protocol, the main signs that radiologists should consider for their reports during their clinical activity and future perspectives.MRI protocol for rectal cancer staging and re-staging.• MRI findings that radiologists should consider for reports during everyday clinical activity.• Perspectives regarding the development of latest technologies.•  Magnetic resonance imaging (MRI) represents the first-choice exam for rectal cancer staging. Usually, clinicians require rectal MRI in case of positive colonoscopy for cancer and/or impossibility to endoscopically remove the lesion. In this context, the role of other imaging modalities, such as endorectal ultrasound and computed tomography, has some limitations. Endorectal ultrasound has the advantage, compared to MRI, of being able to differentiate T1-T2 tumours. However, in case of T3-T4 tumours, it is burdened by its limited field of view, which negatively impacts on the assessment of the mesorectal fat involvement and of tumour extension. Moreover, it is a highly operator-dependent method, requiring a not straightforward learning curve to reach an optimal diagnostic accuracy  leads clinicians to be more demanding with radiologists than in the past, regarding the prediction of the tumour clinical history  Table . Fusco ee et al.  demonstre et al.  showed cConcerning the treatment response prediction with DWI, in the light of good results obtained by using whole-volume tumour segmentations , 43, 44,A potential limitation of DWI is that to date there are no studies showing a conclusive predictive role for the primary staging MRI exam. Concerning this research topic, some studies recently investigated the potential role of perfusion MRI (or DCE-MRI), which allows the analysis of tumour neo-angiogenesis, in terms of pharmacokinetic parameters, using an intravenously administered contrast agent (gadolinium-DTPA or gadolinium-DOTA). The thesis is that, at the primary staging, the angiogenesis of an aggressive tumour would be different from that of a non-aggressive tumour. Moreover, DCE should improve the conspicuity of small residual tumour foci at restaging MRI. For example, Oberholzer et al. , in a lab value (<200 mm2/s) to quantify pseudo-diffusion and high b value (>200 mm2/s) to quantify the true tissue diffusion. In this study, they demonstrated the parameter D was useful to discriminate between good and poor responders.Perfusion MRI is not the only way researchers are following. Due to inconsistent results reported in literature regarding the prediction of treatment response by using ADC values, Nougaret et al.  recentlyAnother exciting topic in the literature regards data analysis providing quantitative features extracted from images by means of a radiomics approach. Three orders of quantitative features can be extracted: first-order features are based on tumour shape, size, intensity and volume; second-order features are generally described as “texture features” and are related to voxel similarity (or dissimilarity) in terms of contrast values; third-order features describe the presence of repetitive or non-repetitive patterns of voxels . All theMagnetic resonance is considered the first-choice technique for the rectal cancer primary staging and restaging after neoadjuvant treatment. Recent recommendations by the European Society of Gastrointestinal and Abdominal Radiology help radiologists to improve their everyday clinical activity with regard to image acquisition, interpretation and reporting. Nowadays, the integration of different imaging modalities, new image analysis approaches and the research of predictive imaging biomarkers represent a promising perspective.


Therefore, the correspondence information for this paper should have appeared as follows:Correspondence to: Dr Hongping Song or Dr Liwen Liu, Department of Ultrasonic Medicine, Xijing Hospital, The Fourth Military Medical University, 172 Chang Le Road, Xi'an, Shaanxi 710032, P.R. Chinasong.hp@foxmail.comE-mail: liuliwen@fmmu.edu.cnE-mail: The authors regret that this this error was not noticed and corrected prior to the publication of the article, and apologize to the readership for any inconvenience caused.


The 177Lu-octreotate therapy has shown promising results in clinical trials by prolonging progression-free survival, but complete responses are still uncommon. The aim of this study was to improve the 177Lu-octreotate therapy by means of combination therapy. To identify radiosensitising inhibitors, two cell lines, GOT1 and P-STS, derived from small intestinal neuroendocrine tumours (SINETs), were screened with 1224 inhibitors alone or in combination with external radiation. The screening revealed that inhibitors of Hsp90 can potentiate the tumour cell-killing effect of radiation in a synergistic fashion . The potential for Hsp90 inhibitor ganetespib to enhance the anti-tumour effect of 177Lu-octreotate in an in vivo setting was studied in the somatostatin receptor-expressing GOT1 xenograft model. The combination led to a larger decrease in tumour volume relative to monotherapies and the tumour-reducing effect was shown to be synergistic. Using patient-derived tumour cells from eight metastatic SINETs, we could show that ganetespib enhanced the effect of 177Lu-octreotate therapy for all investigated patient tumours. Levels of Hsp90 protein expression were evaluated in 767 SINETs from 379 patients. We found that Hsp90 expression was upregulated in tumour cells relative to tumour stroma in the vast majority of SINETs. We conclude that Hsp90 inhibitors enhance the tumour-killing effect of 177Lu-octreotate therapy synergistically in SINET tumour models and suggest that this potentially promising combination should be further evaluated. Following a promising retrospective study  and significantly improved response rates (18% vs 3% after 20 months) in patients with small intestinal neuroendocrine tumours (SINETs), compared with the best standard of care  was aliquoted in DMSO and for in vivo experiments prepared in 5% DMSO and 45% PEG in ddH2O by applying a low amount of heat until dissolved, and was stored at −20°C.The screening library consisted of 1224 inhibitors . The library was batched in 96-well plates and stored at −80°C. The plates were subjected to no more than five freeze-thaw cycles. For 0,  Tyr3]-octreotate with 177Lu and subsequent thin-layer chromatography quality control was performed as previously described .Radiolabelling of [DOTA3 polystyrene phantom. The height of the media in each well was 2 mm and the thickness of the cell layer was approximately 20 µm. The cell layer was positioned at a height corresponding to the distance for the dose maximum of the radiation beam. Irradiation was given at a 180° gantry angle (upwards irradiation), the source surface distance was 900 mm. The radiation field at the isocenter (1000 mm) was 200 × 200 mm2.External radiation was given using a Varian TrueBeam linear accelerator with 6-MV nominal photon energy and a dose rate of 8.2 Gy/min absorbed dose to water. During irradiation, cell culture plates were held in a 220 × 220 × 90 mmFor all experiments, 50,000 cells were seeded onto black non-optical 96-well cell culture plates  and 24 h was allowed for cell attachment before the start of the experiment. At the end of all experiments, the cell viability was estimated using alamarBlue (Thermo Fisher Scientific), a fluorescence-based assay for measurement of the reducing capacity of metabolically active cells. All assay plates were incubated for 6 h at 37°C, and were then analysed with a fluorescence plate reader .N = 8). In addition, each experiment included one cell-free plate for background subtraction (N = 96) and one DMSO-treated plate as vehicle control (N = 96). The screening library was added to a final concentration of 1 µM. After adding the inhibitor library, combination-treated cells were subjected to 3 Gy external radiation within 30 min. Experiments were repeated two or three times for inhibitors only, for radiation only and for combination treatment. Screening experiments were ended after 48 h. For cell line dose-response experiments with ganetespib, GOT1 and P-STS were treated with ganetespib at indicated doses for 72 h before plates were analysed. Each dose included four replicates and the experiment repeated three times. For experiment with patient-derived tumour cells, first-passage cell cultures were generated from eight patients. The cells were treated with radiation , ganetespib  or both treatments combined . For each patient and treatment the experiment included 12 replicates and was performed once due to the limited cell numbers. Cell plates were analysed 72 h after the start of the experiment.For screening experiment, the inhibitor library was added to 96-well culture plates, with each plate also including internal DMSO controls (N = 6), 15 MBq 177Lu-octreotate (N = 5), 50 mg/kg ganetespib (N = 5) or a combination of both treatments (N = 5). These amounts, and the single-administration design, were chosen in order to obtain a moderate but clearly measurable treatment effect of each monotherapy. Mouse tumours were measured twice weekly with slide callipers. Growth inhibition was calculated as the relative tumour volume compared to vehicle-treated mice at a given time point. Tumour regression was calculated as the relative tumour volume compared to treatment start. Water and autoclaved food were available ad libitum. After 14 days the experiment was ended and mice sacrificed by intraperitoneal injection of 60 mg/mL pentobarbital , followed by cardiac puncture.GOT1 tissue was transplanted subcutaneously to BALB/c nude mice  as previously described . All samples and chromogranin A standards were analysed using two technical replicates.The tissue microarray was generated from patients who underwent surgery for SINETs at Sahlgrenska University Hospital in the years 1986 to 2013. Details of the construction of the tissue microarray have been described previously . Tissue blocks from patient tumours were obtained from Sahlgrenska University Hospital. These blocks were prepared for routine clinical histopathology. Tissue microarrays were generated as described above.lower, equal or higher. The overall Hsp90 expression was evaluated in 767 SINETs from 379 patients and based on the staining intensity of tumour cells scored as ‘1’ or ‘2’. The Ki67 index was calculated by manually counting the percentage of labelled tumour cell nuclei on printed images (Sections (3–4 μm) from paraffin blocks were placed on glass slides and treated in Dako PT-Link using EnVision FLEX Target Retrieval Solution (high pH). The following primary antibodies were used: anti-chromogranin A , anti-synaptophysin , anti-5HT , anti-pan-cytokeratin , anti-SSTR2A , anti-Ki67  and anti-Hsp90 . Immunohistochemical staining was performed in a Dako Autostainer Link using EnVision FLEX according to the manufacturer’s instructions (DakoCytomation). EnVision FLEX+ (LINKER) rabbit or mouse was used for all stainings except anti-chromogranin A (PHE5) and anti-pan-cytokeratin. Positive and negative controls were included in each run. Tumour regression was evaluated by board-certified pathologist (O.N.) and scored according to We obtained approval from Regional Ethical Review Boards in Gothenburg, Sweden, for the use of clinical materials for research purposes and for all animal procedures. Consent has been obtained from each patient after full explanation of the purpose and nature of all procedures used.P < 0.05) was assessed using Fisher’s exact test. The FDRs for the overrepresented inhibitor classes were calculated as above. The analysis of the longitudinal in vivo experiment was done based on the log-transformed relative tumour size of xenografted tumours. A linear mixed model was fitted to tumour volume measurements for each treatment  with a random slope parameter modelling the growth rate. Synergy between treatments was assessed by comparing the effect size (slope) of the combined treatment with the sum of the effect sizes from the individual treatments. Significance was calculated using a Z-test with effect sizes and standard errors estimated by the linear mixed models. Any P value less than 0.05 was considered statistically significant. Comparisons of regression score (Becker score) between treatment groups was done using the Mann–Whitney nonparametric test. The analysis of the cell viability data from the experiments with patient-derived tumour cells was done as follows. Technical replicates were averaged and relative intensities, in comparison to the control, were calculated. For each radiation level , a linear model was used to estimate the trend in relation to the log-transformed concentration of ganetespib. The difference in slope between radiation and vehicle control was used to assess synergy. Any P value less than 0.05 was considered statistically significant. Survival analysis, testing the difference in overall survival from time of surgery between high and low expression of Hsp90 was performed using Kaplan–Meier estimator with log-rank test. For patients with multiple tumours (more than one tumour site) only one tumour was included with the following priority: primary tumour, lymph node metastasis and hepatic metastasis. The survival analysis using the full patient cohort consisted of 312 patients and the 177Lu-octreotate-treated cohort consisted of 43 patients.For each cell line, inhibitor fluorescence intensities generated by the cell viability assay were log-transformed and normalised to a common scale. For each inhibitor, the interaction between radiation and inhibition was analysed using a two-way ANOVA with an interaction term. Significance was calculated based on the size of the interaction term. The false discovery rates (FDRs) were calculated using the Benjamini–Hochberg algorithm . Overrep177Lu-octreotate therapy in a synergistic manner, we screened cell lines GOT1 and P-STS using an inhibitor library consisting of 1224 inhibitors, both as single agents and in combination with external radiation. We were able to identify 28 and 37 inhibitors, respectively, in screenings of GOT1 and P-STS cells that significantly synergised with radiation (P < 0.05) in killing tumour cells in vitro , with 11 out of 18 inhibitors having a significant synergistic effect (50 = 7.2 Gy vs not reached) and GOT1 was more sensitive to ganetespib compared to P-STS at all tested concentrations  . The combination therapy had the overall best response, leading to 42% tumour regression compared with treatment start after 10 days. To determine whether there was synergy between ganetespib and 177Lu-octreotate in reducing tumour volume, we performed a longitudinal analysis using a linear mixed-model analysis applied over time points with sustained treatment effect  after excluding one extreme individual in the ganetespib monotherapy group with a value >3.5 standard deviations from the mean (otherwise P = 0.13). The extreme individual belonged to the ganetespib-treated group and had a tumour which regressed 48% after 14 days. The other ganetespib-treated mice had tumours that instead progressed, with an average tumour growth of 55%.The tumour volume of all mouse tumours were measured twice weekly and the experiment was ended after 14 days  . For all eight individual patient tumours, there was a consistent trend towards synergy , antagonistic (a response that is less than additive) or synergistic (a response that is more than additive). When combining treatments, it is advantageous to achieve a synergistic interaction, as this could increase the therapeutic index while reducing toxicity .


Fluid overload (FO) is associated with unfavorable outcomes in critically ill children. Clinicians are encouraged to avoid FO; however, strategies to avoid FO are not well-described in pediatrics. Our aim was to implement a bundle strategy to prevent FO in children with sepsis and pARDS and to compare the outcomes with a historical cohort.A quality improvement initiative, known as preemptive fluid strategy (PFS) was implemented to prevent early FO, in a 12-bed general PICU. Infants on mechanical ventilation (MV) fulfilling pARDS and sepsis criteria were prospectively recruited. For comparison, data from a historical cohort from 2015, with the same inclusion and exclusion criteria, was retrospectively reviewed. The PFS bundle consisted of 1. maintenance of intravenous fluids (MIVF) at 50% of requirements; 2. drug volume reduction; 3. dynamic monitoring of preload markers to determine the need for fluid bolus administration; 4. early use of diuretics; and 5. early initiation of enteral feeds. The historical cohort treatment, the standard fluid strategy (SFS), were based on physician preferences. Peak fluid overload (PFO) was the primary outcome. PFO was defined as the highest FO during the first 72 h. FO was calculated as (cumulative fluid input – cumulative output)/kg*100. Fluid input/output were registered every 12 h for 72 h.p < 0.01] and maintained during the study. These differences were due to less fluid input (MIVF and fluid boluses). There were no differences in the renal function test. PRBC requirements were lower during the first 24 h in the PFS (5%) compared to SFS . MV duration was 81 h  in PFS and 118 h (85154) in SFS(p < 0.05). PICU LOS in PFS was 5  and in SFS was 8  days.Thirty-seven patients were included in the PFS group ) and 39 with SFS ). PFO was lower in PFS (6.31% [IQR4.4–10]) compared to SFS (12% [IQR8.4–15.8]). FO was lower in PFS compared to CFS as early as 12 h after admission [2.4 v/s 4.3, Implementation of a bundle to prevent FO in children on MV with pARDS and sepsis resulted in less PFO. We observed a decrease in MV duration and PICU LOS. Future studies are needed to address if PFS might have a positive impact on health outcomes.The online version of this article (10.1186/s12887-018-1188-6) contains supplementary material, which is available to authorized users. During the last two decades, intravenous fluid administration has been the cornerstone of treatment of children with hemodynamic instability. Restoration of circulating blood volume and perfusion to tissues is the primary end point of this therapy, decreasing mortality and morbidity of critically ill children with clinical signs of poor perfusion and shock . SupportDespite these overwhelming data, few studies in pediatrics have addressed how to avoid significant fluid overload in patients under MV.The objective of this study was to describe the implementation of a preemptive fluid strategy in children with sepsis and ARDS. In addition, a comparison with an historical cohort of infants was made.Expedited review by the IRB approved the quality improvement project, waiving the requirement for a written consent.Setting: During a 12-month period (January to December 2016), all children admitted to the Pediatric Intensive Care Unit at Hospital Padre Hurtado were screened. Our unit is a 12-bed general medical and surgical PICU and does not take care of patients after congenital heart surgery or those receiving a transplant.Participants: All patients younger than 24-months who received MV and fulfilled the pediatric ARDS (pARDS) and sepsis criteria, were prospectively identified. pARDS was defined according to the PALICC criteria  and sepsMaintenance of intravenous fluids (MIVF) at 50% of baseline requirements estimated by the Holliday-Segar formula, ensuring adequate glucose infusion rate for normoglycemia.Preparation of continuous infusions of medications (sedation and vasoactive drugs) and intravenous treatments  concentrated to the minimum volume recommended. Use of miniaturized devices for hemodynamic monitoring  at the minimum rate of saline infusion.Use of dynamic preload markers (pulse pressure variation) in addition to clinical assessment to decide administration of fluid boluses and early titration of vasoactive drugs.Consideration of early use of diuretics when hypovolemia was ruled out, resuscitation goals were met and urinary output was less than 0.5 ml/kg/h.Early initiation of enteral feeds.As previously reported , this prA Historical cohort data was collected retrospectively with the same inclusion/exclusion criteria from June 2014 to December 2015. These patients were treated with standard fluid strategy and compared with the prospectively collected data of the patients managed with PFS.Resuscitation phase: hemodynamic resuscitation based on Surviving Sepsis Campaign , 3.Stabilization phase: patients received MIVF at 100% of baseline requirements estimated by the Holliday-Segar formula.Depletive phase: Initiation of diuretics after meeting resuscitation end points, hemodynamic stability and absence of any marker of dysoxia.Standard fluid strategy can be summarized as:Demographic and clinical data were recorded in a relational database. Fluid intake and output (I/O) were recorded every 12 h during the first 3 days after admission. To determine the causes of fluid overload and variations in clinical practice after initiation of the protocol, fluid intake was divided into MIVF, fluid bolus, enteral feeds, drug administration  and packed red blood cells (PRBC) transfusions. Renal function was monitored daily and electrolyte alterations were monitored every 12 h or more frequently depending on the treating physician’s assessment. Urinary output and diuretic use were also recorded.Vasoactive drug (VAD) support was recorded and maximum vasoactive inotropic score (VIS) score was calculated daily . The perThe primary outcome was to measure peak fluid overload (PFO). Fluid input and output were recorded as secondary outcomes to determine the causes of FO. Other clinical outcomes such as duration of MV, hospital and PICU length of stay (LOS) were recorded.P-value less than 0.05 indicating statistical significance. The SPSS software package  was used for the statistical analyses.Descriptive statistics were used to summarize all continuous and categorical variables. Comparisons between patient groups were performed using Fisher’s exact test for categorical variables and the Mann-Whitney U test for continuous variables because of concerns about the normality of the distribution of these variables. Two-way repeated measures ANOVA test was performed for comparisons (fluid overload) among and between groups during the study period. All statistical tests were 2-sided and were performed with a Thirty-seven patients were included in the PFS group and compared with 39 patients in the SFS group Table . No mortP < 0.05) . The SFS group required more frequent continuous infusions of diuretics at 48 and 72 h.  and VIS was significantly lower during the first and the third day of admission in the PFS group. Use of greater than or equal to 2 vasoactive drugs was more frequent in the SFS group during the third day after admission . Additionally, PICU LOS was shorter in the PFS group compared to the SFS group .Time to successful extubation was 81.5 h [IQR 56.5–100.5] in the PFS group and 118 h [IQR 85.5–154.5] in the SFS group  over the first 48 h can be easily applied in most PICU and may prevent significant FO.Future collaborative studies are needed to address if a preemptive fluid strategy after resuscitation from shock, in addition to optimal care for pARDS  might have a positive impact in outcomes.Additional file 1:Figure S1. Vasoactive drug use in standard fluid strategy and preemptive fluid strategy at day 1, 2 and 3 of study. * P < 0.05. Abbreviations: PFS: preemptive fluid strategy; SFS: standard fluid strategy. (JPG 226 kb)


Endobronchial ultrasound has revolutionized the field of bronchoscopy and has become one of the most important tools for the diagnosis of intrathoracic lymphadenopathy and para-bronchial structures. The reach of this technique has not been limited to these structures and pleural lesions have been at times accessible. To our knowledge, pleural fluid collections have not been accessed with endobronchial ultrasound (EBUS).52-year-old women with dyspnea, fever and a new loculated pleural effusion that was suspected to be the source of the fever but was not accessible through traditional thoracentesis.Malignant pleural effusion.Sampling and drainage of the loculated pleural fluid collection using EBUS scope introduced via the esophagus.Infection excluded. Resolution of fever and improved dyspnea after drainage of effusion.The convex curvilinear ultrasound bronchoscope allows unprecedented access to thoracic structures. The reach is not limited to mediastinal lymph nodes and parenchymal masses adjacent to the airways, and pleural space and pleural fluid are at times accessible, particularly when one considers the esophageal approach. We present a case of a loculated fluid collection that was suspected to cause fever and dyspnea and was not amenable to traditional thoracentesis, but was successfully sampled and drained using the EBUS scope via the esophagus.Since its introduction in 2004, endobronchial ultrasound-transbronchial needle aspiration (EBUS-TBNA) has become one of the most important tools available to chest physicians for the diagnosis of intrathoracic lymphadenopathy and para-bronchial structures.2A 52-year-old woman with a history of stage 4 breast cancer presented to the hospital for fever of 1-week duration and worsening shortness of breath. She had known pleural involvement of her cancer and underwent tunneled pleural catheter placement 8 months prior due to recurrent malignant pleural effusion. She achieved spontaneous pleurodesis and the catheter was eventually removed without significant residual fluid collection seen on imaging. Upon our exam, the prior tunneled catheter site was nonerythematous and clearly not infected. The chest x-ray; however, showed a new left apical well-rounded opacity. Subsequent chest computed tomography scan showed a new 6.5 cm well-circumscribed left apical lesion of homogeneous density consistent with fluid as opposed to a mass  has revolutionized the field of bronchoscopy and has become one of the most important tools available to chest physicians for the diagnosis of intrathoracic lymphadenopathy, para-bronchial structures and the staging of lung cancer.,9 This is of real concern when puncturing a pleural fluid collection. The postulated mechanism for pleural inoculation is that as the bronchoscope traverses the naso-oropharyngeal region, the working channel becomes contaminated. When the transbronchial needle passes through the working channel, the needle becomes contaminated and could potentially inoculate sampled tissue. Although the needle designed for this scope has an outer sheath to minimize sample contamination, this sheath still passes through the working channel. When the needle is deployed at the site of interest, it passes through the distal end of this sheath and can become contaminated. Another possibility is contamination of the stylet when multiple punctures are applied. To prevent these complications, universal guidelines should be followed including disinfection of devices according to the guidelines and careful observation of the post-procedure course for signs of infection are important. Particular attention should be given to avoid stylet contamination. Prophylactic antibiotic administration, although not recommended in routine diagnostic bronchoscopy, could be considered when puncturing fluid collections and cystic lesions. In our case, in addition to our patient being on antibiotics, attention was paid to stylet manipulation and the number of punctures was limited.A legitimate concern is the risk of introducing an infection in the pleural space. EBUS-TBNA has been shown to be a safe procedure with very low infectious complications rate. Most infectious complications were reported when the puncture site is necrotic or cystic, where the blood flow is slight, resistance is decreased, and bacterial attachment clearance is decreased.In conclusion, the convex curvilinear ultrasound bronchoscope allows unprecedented access to thoracic structures. These structures are not limited to mediastinal lymph nodes and parenchymal masses adjacent to airways, and pleural space and pleural fluid are at times accessible. Additionally, an esophageal approach to making the diagnosis sometimes offers opportunities not permitted via the airways, either due to anatomy or greater patient risk. We hope that this case provides an insight into the utility of this tool in the hands of the interventional pulmonologist.Conceptualization: Steven Cocciardi, Ziad Boujaoude.Data curation: Steven Cocciardi, Ziad Boujaoude.Formal analysis: Ziad Boujaoude.Writing – original draft: Steven Cocciardi, Ziad Boujaoude.Writing – review and editing: Amit Borah, Rocco Terrigno, Wissam Abouzgheib, Ziad Boujaoude.


The EUCLID study group reports remarkably excellent outcome in children with community acquired septic shock. Successful management of pediatric septic shock is challenging because not all children require the same therapies at presentation, and individual patients require changing therapies over time. The pathway to success is not a ‘one size fits all’ approach. Health care systems and practitioners must be both quick and nimble. Quick because untreated bacterial infections can double in pathogen numbers every 28 minutes , 2 and uThe likelihood of success increases with a layered systems level approach. First, the hospital must provide practitioners with resources that facilitate use of time-sensitive septic shock recognition trigger tools and a concomitant one hour–three element bundle that 1) attains a blood culture and gives empiric intravenous antibiotics, 2) gives a 20 mL/kg bolus(es) of intravenous isotonic fluid if there is no hepatomegaly or crackles, and 3) gives a peripheral epinephrine infusion if shock is fluid refractory . Next, tFollowing initial resuscitative studies demonstrating the benefit of fluid resuscitation and antibiotics in the first hour after identification of septic shock , individ2 and < 6.0 L/min/m2 and systemic vascular resistance index > 800 dynes/s/m2 and < 1200 dynes/s/m2, 4) normal mean arterial pressure minus central venous pressure (MAP − CVP) = 55 + 2 × age mm Hg, 5) central venous oxygen saturation (ScVO2) > 70%, and 6) even fluid balance after the initial fluid resuscitation period.In addition to trigger tool-facilitated recognition and administration of antibiotics and a 20 mL/kg fluid bolus(es) within an hour, further administration of antibiotics, fluid, and hemodynamic therapies in the emergency department and intensive care unit were guided by multimodal monitoring using a personalized approach that maximized benefits while reducing risks of applying therapies without regard to individual pathophysiology and response . The recStreptococcus pneumoniae were most common, with the latter being associated with increased morbidity and mortality; however, in the US Staphylococcus aureus and methicillin-resistant S. aureus in particular are associated with increased mortality. Culture and sensitivity analyses should be attained as infections differ among individuals. When cultures dictate, empiric antibiotics should be narrowed to the most sensitive antibiotic given at a dosage above the minimum inhibitory concentration (MIC) needed to kill the microbial pathogen. Source control is particularly important in the presence of an abscess or necrotic tissue nidus. Suspicion for and surveillance of nosocomial infection is also warranted. Interestingly, improved outcome with use of peripheral epinephrine infusion compared to peripheral dopamine in patients with fluid refractory shock is associated not only with faster shock reversal but also with a significant reduction in nosocomial infection [Empiric antibiotics and source control differ according to context. In the EUCLID study in Europe, meningococcemia and nfection .2 and systemic vascular resistance index > 800 and < 1200 dynes/s/m2 [Children who present with community acquired septic shock are more likely to need inotropic support (epinephrine) for cardiac dysfunction than children who have hospital acquired septic shock who are more likely to require vasopressor support for vascular dysfunction (norepinephrine) . Howevernes/s/m2 .2) > 70% [Central venous pressure alone has limited utility for documenting preload since it is not consistently associated with intravascular volume and may be dynamically affected by therapies such as positive pressure ventilation and the effects of diastolic dysfunction. The goal of therapy is not to achieve a CVP target, but rather to increase perfusion pressure (MAP − CVP) to achieve adequate urine output, distal perfusion, and improved tissue oxygenation. Two studies reported improved survival from septic shock when targeting normal MAP − CVP for age and central venous oxygen saturation (ScvO2) > 70% , 13. Ano2) > 70% .Separate from appropriate personalized fluid resuscitation in the first hour, administration of fluid beyond the polyuria reserve capacity of the kidney is common in children on mechanical ventilation longer than 24 h related in part to receipt of medications diluted in carrier volumes amounting to more than 30 mL/kg/day . AttentiMortality from pediatric septic shock is remarkably low when managed in hospitals that facilitate use of an early recognition trigger tool with a three-element bundle followed by a personalized medicine approach Fig. .Fig. 1Us


Microbial community profiling showed that the relative abundance of known lignocellulose-degrading bacteria corresponded to lignocellulose-degrading enzymatic activities. Some dominant indigenous cellulolytic and hemicellulolytic bacteria in seed sludge  and rumen fluid  became undetectable or markedly decreased in abundance in the fermenters fed rumen fluid-treated rapeseed, whereas some bacteria derived from seed sludge  and rumen fluid  remained detectable until the completion of methane production. Thus, several lignocellulose-degrading bacteria associated with rumen fluid proliferated in the fermenters, and may play an important role in the degradation of lignocellulosic compounds in the fermenter.The pretreatment of lignocellulosic substrates with cattle rumen fluid was successfully developed to increase methane production. In the present study, a 16S rRNA gene-targeted amplicon sequencing approach using the MiSeq platform was applied to elucidate the effects of the rumen fluid treatment on the microbial community structure in laboratory-scale batch methane fermenters. Methane production in fermenters fed rumen fluid-treated rapeseed (2,077.3 mL CH We previously investigated the preservation of cattle rumen fluid for application to methane production  and its potential for the pretreatment of lignocellulosic substrates, and the findings obtained revealed that the rumen fluid treatment improved the degradation rates of cellulose, hemicellulose, and lignin and achieved 1.5–3.2-fold greater methane production than non-treatment systems . However, if added rumen microbes rapidly die out within the methane fermenter, they are applicable during only the first (hydrolytic) phase in two-phase (hydrolytic phase+methanogenic phase) biogas digesters.Lignocellulosic biomass, composed of cellulose, hemicellulose, and lignin, is the most abundant natural polymeric carbon source in the world  and, thuoduction   of methane fermenters fed rumen fluid. The present study monitored time-dependent changes in the microbial community structure and investigated the fate of rumen-derived lignocellulose-degrading bacteria and indigenous lignocellulose-degrading bacteria of methane seed sludge during methane fermentation.The present study focused on lignocellulose-degrading bacteria and methanogenic archaea that contribute to the methanogenesis of lignocellulose and investigated the impact of adding rumen fluid containing an endogenous microbial community on the indigenous bacteria of methane seed sludge as well as the fate of the added rumen microbial community via MiSeq next-generation sequencing during methane fermentation. Previous studies , 34, 41 Brassica napus) were air-dried and milled to produce lignocellulosic biomass that was used as the substrate for methane production after the pretreatment with rumen fluid.The stems and leaves of rapeseed  to remove coarse solids, and stored at 37°C. Prepared rapeseed (9.0 g dry weight) was pretreated with 300 mL of rumen fluid at 37°C on a rotary shaker at 170 rpm for 6 or 24 h. The rumen fluid treatment was performed in duplicate. The care and use of animals in the present study were approved by the Institutional Animal Care and Use Committee of Tohoku University.Methane seed sludge for the initiation of the experiment was prepared using anaerobically digested sludge collected from the biogas plant at Tohoku University . Some substrates  were the4, CO2, and H2) concentrations were evaluated using a gas chromatograph , volatile fatty acid (VFA) concentrations were measured by a high-performance liquid chromatograph , and chemical oxygen demand (COD) was assessed using a colorimetric method based on Cr, as previously described (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to confirm the closest species. All sequence data were deposited in the DRA of the DDBJ database under DRA008816.The biomass chemical composition was analyzed by the Agricultural Product Chemical Research Laboratory in the Tokachi Federation of Agricultural Cooperatives using the detergent fiber method  and estiescribed . Lignoceescribed . DNA extescribed . TaxonomThe chemical composition of rapeseed was as follows: 39.4% non-fibrous carbohydrate, 21.7% cellulose, 3.3% hemicellulose, 3.2% lignin, and other components (such as protein and ash). The TS and VS compositions of rapeseed were 91.9 and 74.2%, respectively. The TS and VS of rumen fluid after filtration were 1.6 and 0.8%, respectively.e.g., cellulose, hemicellulose, and lignin)  than from untreated control rapeseed , which is consistent with previous findings  . Batch-t lignin) . More meAfter quality filtering, paired-end read assembly, quality control, and chimera removal, 2,663,094 V3–V4 16S rRNA sequence reads  were obtained from raw MiSeq data from 21 samples. The average sequence read length after primer subtraction was 450.9 bp. With a subsample of 46,723 reads per sample, rarefaction curves  demonstrSynergistetes and WWE1 increased over time. Aminobacterium was the main member of the phylum Synergistetes in the methane fermenter fed untreated rapeseed, whereas Thermovirgaceae, vadinCA02, HA73, and Aminobacterium were the main members in the fermenter fed with rumen fluid-pretreated rapeseed -degrading bacteria    was performed , and the results obtained indicated that the composition in the fermenter mixed with pretreatment fluid did not return to that in the seed sludge. Alpha diversity indices  also indicated that species richness increased due to the addition of pretreatment fluids   was identified by a homology search of the GenBank database using BLAST. Nine OTUs that are phylogenetically related to known cellulolytic, xylanolytic, and aromatic-degrading bacteria were detected in the methane fermenter without the pretreatment  activities. Regarding the xylan-degrading population, changes in the relative abundance of OTU 572010, associated with known xylanolytic bacteria , mirrorteroides ), mirrorlibacter , Prevoteevotella , Celluloilyticum , and Rumnococcus ) mirroreorhabdus ; howevertibacter , Acinetotobacter , and Pseudomonas )  was added to the methane fermenter, and the impact of the addition of this fluid on the above-mentioned indigenous lignocellulose-degrading bacteria of methane seed sludge . OTU14124, 342612, 572010, and 768947 were associated with known cellulolytic and xylanolytic bacteria   and declibacter ), increaudomonas ) became hohabdus , and Acitobacter ) in thisi.e., not detected in methane seed sludge) are shown in i.e., Ruminococcus albus [Ruminococcus, and 4 OTUs were sequences associated with R. albus and remained detectable until the completion of methane production . These methanogenic archaea obtain energy through the reduction of methanol or CO2 to CH4 using H2 as a substrate  is generally required . Sequenoduction . Althougnivibrio ). OTU842ubstrate . To achirequired , 35. Furrequired . To facihanogens . The relhobacter , 15), sli.e., members of Butyrivibrio [Prevotella [Pseudobutyrivibrio [Some of the OTUs  in rivibrio , Prevoteevotella , 33, andrivibrio ), which i.e., R. albus) remained detectable until the completion of the methane production process. Conversely, indigenous bacteria associated with known cellulolytic and hemicellulolytic bacteria  in methane seed sludge markedly decreased after the addition of the pretreatment fluid. In addition, a rumen-derived archaeon associated with known H2-utilizing methanogenic archaea  also remained detectable until the completion of the methane production process after the addition of pretreatment fluid. Thus, the methane fermenter in the present study became a unique fermenter composed of pretreatment fluid-derived cellulolytic, hemicellulolytic, and methanogenic rumen microbes and indigenous species in methane seed sludge. Differences in the species composition of the methane fermenter suggested by PCoA may be caused by these rumen microbes that survived. If these pretreatment fluid-derived rumen microbes persist in the methane fermenter, they are expected to enhance lignocellulose-based methane productivity. Future studies are needed to elucidate whether these microbes directly contribute to the enhanced efficiency of methane fermentation by confirming the presence of enzyme mRNA  in pretreatment fluid-derived rumen microbes within the methane fermenter.The present study demonstrated that rumen-derived bacteria associated with known cellulolytic and hemicellulolytic bacteria (2-utilizing methanogenic microbes introduced into the fermenter via the pretreatment fluid inoculation remained detectable until the completion of methane production. Thus, these results showed that the methane fermenter in the present study is a unique fermenter composed of pretreatment fluid-derived rumen microbes and indigenous species in methane seed sludge and also that this microbial community structure may contribute to methane productivity.The rumen fluid pretreatment enhanced methane productivity. The microbial community structure analysis demonstrated that dominant indigenous cellulolytic and hemicellulolytic bacteria in methane seed sludge no longer increased or markedly decreased in relative abundance after the addition of the pretreatment fluid. Conversely, certain rumen-derived cellulolytic, hemicellulolytic, and H