Our result highlighted that using palatable fluids as a strategy to increase fluid consumption during high-intensity gameplay in the heat may not be effective if used without carbohydrates and electrolytes. Practitioners could consider both fluid palatability and composition in establishing a hydration plan for athletes.Using palatable fluids to enhance drinking in athletes who display insufficient compensatory hydration behaviour may mitigate the risks of hypohydration and performance deficits. However, it is unclear whether flavour can independently enhance fluid consumption. This study examined the effects of a colourless, artificially sweetened flavoured water (FW), without carbohydrates and with negligible amounts of sodium, compared to plain water (W) on fluid consumption in male collegiate basketball players in a practical game setting. Eighteen male basketball players (age 23.1 ± 1.3 years) played a 3v3 basketball small-sided game. The players were randomly assigned to consume either FW or W. Pre-game urine-specific gravity, fluid consumption, body mass, and hedonic taste perceptions were assessed. Basketball performance was analysed through notational analysis. Ratings of perceived exertion and thirst were recorded at pre-, post-game, and at each rest period. Heart rate was recorded throughout the gameplay. Despite significantly higher hedonic ratings for FW than W (6.78 ± 0.83 vs. 5.56 ± 1.33,  Optimal hydration plays a key role in the health and sport performance of athletes. While optimal fluid balance is dependent on a multitude of factors, it is conventionally defined as the avoidance of body water deficits greater than 2–3% of body mass while preventing excessive fluid consumption  . During Team sports are often characterized by intermittent bursts of high intensity work which can elicit significant sweat losses . StudiesFluid needs are highly individualistic, and achieving an optimal level of hydration for performance can be complex. Establishing a hydration plan that accounts for the individual player in the context of the environment and sport may be effective in developing strategies to optimise their hydration needs . SpecifiFluid palatability, defined as the hedonic evaluation of sensory factors, such as taste and smell, is a major determinant of fluid consumption ,7. To elResearch on fluid palatability and fluid consumption in hot-humid environments has been predominantly conducted in a laboratory setting  and performed at relatively low exercise intensities ,16,17. FThe aim of this study was to examine if fluid palatability that is independent of fluid composition can enhance voluntary fluid consumption in male collegiate basketball players in a practical game setting. Though we focused primarily on examining hydration behaviour, we also sought to address the lack of information available on the physiological demands and fluid needs of small-sided games (SSG) . Research on 3 × 3 basketball remains scarce despite its growing popularity, which culminated in its inclusion in the 2020 Tokyo Olympics, as well as the increased use of SSG in light of the COVID-19 outbreak ,19,20. TEighteen male basketball players were recruited via convenience sampling from a national varsity league in Singapore. All players were tropical natives who lived and trained in a hot and humid country. Players presented with no injury and/or illness, and were excluded from the study if they reported to be on any form of medication and/or had consumed caffeine, alcohol, or diuretics prior to the test session. Institutional ethical approval for the study was obtained (IRB-2019-06-041-01). Demographic data are in We adopted a randomised between-subject design. The small-sided basketball game involved six players (3v3) on a full-court with an active playing time of 30 min. Prior to gameplay, the players were randomly assigned to consume either a colourless, flavoured water  (FW) or plain water (W). Hydration status and hedonic taste perceptions were assessed prior to and after gameplay. Fluid consumption was determined post-game. Heart rate responses and rating of perceived exertion (RPE) were measured to assess the physiological load. Basketball performance indicators were assessed by notational analysis.Environmental temperature and humidity at the start of each SSG was measured using a calibrated handheld environmental meter . Mean environmental temperature and humidity was 31.7 ± 0.5 °C, 62 ± 4%.Prior to gameplay, all players performed a standardised warm up protocol. The structure of the SSG involved six players (3v3) on a full-court. The total duration of the gameplay was 40 min, which consisted of three periods of 10-min active gameplay interspersed by two 5-min rest periods, where players returned to their designated rest area. Each 10-min period of active gameplay involved two 5-min halves, with a 30-s rest at half-time for the teams to swap ends of the field of play. The gameplay was officiated by a referee. The overview of the game structure is displayed in This study used an SSG instead of a traditional 5 × 5 or 3 × 3 basketball game, as SSG are commonly used in basketball training to build up physical fitness and improve technical skills ,24. SpecEach player was randomly assigned either the FW or W condition, and was provided with two 1500 mL bottles containing their assigned fluid. Each bottle was weighed to the nearest ±0.01 kg on a digital scale . Both fluids were commercially available and packaged in similar transparent bottles. The players were blinded to the brand identity of the fluids. The FW was lemon-flavoured, artificially sweetened with acesulfame K and sucralose, and contained negligible amounts of sodium (<10 mg per 250 mL). Both acesulfame K and sucralose can improve fluid palatability, as they enhance sweetness and mask unpleasant bitter and sour tastes, respectively . As coolA sensory questionnaire was used to measure the perceived intensities of sweetness, saltiness, and sourness . Each sePre-game hydration status was assessed on urine-specific gravity (USG) upon arrival using a clinical refractometer . Two USG thresholds were used to determine hypohydration: (i) USG > 1.020 and (ii) USG > 1.025. As the conventional limit of 1.020 has been shown to incorrectly classify athletes as hypohydrated when their blood serum osmolality indicates euhydration , a secondary threshold of 1.025 was included to increase the specificity of USG to detect hypohydration .Post-game hydration status was assessed through percentage changes in body mass. Voided body mass was measured at pre- and post-game, where players towel-dried and wore their undergarments only. Body mass was measured to the nearest 0.01 kg on a digital scale .Fluid consumption was determined from the difference between the initial mass of the bottle and the final mass of the bottle post-game. Bottle mass was measured to the nearest ±0.01 kg using a digital scale . The players were allowed ad libitum drinking throughout gameplay, and no attempt was made to influence the drinking behaviours of the players. The total fluid consumption for each player was calculated using the following formula :Fluid coTo calculate sweat rate, urine output was collected and weighed. Though the players were specifically told that they could visit the toilet at any point throughout gameplay, none of them did so. Post-game urine output was collected and weighed. The total testing duration for all the players was two hours. The sweat rate for each player was calculated using the following formula :Body masRatings of perceived exertion (RPE) was recorded at pre-game, during gameplay (10- and 25-min mark), and at post-game (40-min mark) .Thirst was evaluated concurrently at pre-game, during gameplay (at 10- and 25-min mark), and at post-game (40-min mark) using a 9-point scale .All players were fitted with a heart rate monitor , and heart rate was monitored throughout the session.To assess basketball performance, statistics for the number of assists (AST), defensive rebounds (DR), and two-point and three-point field goal percentages  were recorded through notational analysis . These pp < 0.05 with 95% confidence intervals. Results are expressed as mean ± SD or frequencies, unless otherwise stated. The independent samples t-test was performed to examine significant between-group differences. Effect sizes were computed, and thresholds were set at 0.2—small, 0.5—medium, and 0.8—large [Data analysis was conducted using SPSS Statistics, version 23 , with statistical significance set at .8—large . Where t.8—large , which p.8—large . When thp = 0.170). The demographic information and pre-game hydration status of the players are displayed in At baseline, there were no differences between the pre-game USG of both groups (p = 0.033). However, there were no significant differences in fluid consumption (p = 0.068) and body mass changes (p = 0.077). MBD analysis highlighted that fluid consumption was likely higher in W compared to FW, with likely lower body mass losses in FW (−0.941 ± 0.524) compared to W (−0.534 ± 0.376). The palatability ratings and hydration status are shown in Hedonic ratings were significantly higher for FW (6.78 ± 0.83) than W (5.56 ± 1.33)  in plasma osmolality activates the osmotic thirst mechanism . Hence, An interesting observation in this study is the non-corresponding findings between hedonic value and fluid consumption. Despite the significantly higher hedonic values reported for FW, fluid consumption was likely higher in W. Consequently, the players who consumed W experienced slightly lower levels of dehydration. Though the underlying mechanisms behind this were unclear, there was a possibility that FW may decrease voluntary drinking. From an applied perspective, the non-corresponding findings may indicate that in addition to assessing athletes’ fluid preferences, it may also be meaningful to observe their on-field drinking behaviours.In the present study, hydration status did not influence physiological responses and basketball performance as assessed by notational analysis. Though the players who consumed W experienced slightly lower levels of dehydration, there were no differences in physiological responses or basketball performance between both conditions. All the players appropriately gauged fluid consumption throughout gameplay, and body mass changes were within the 2% threshold. This self-regulatory drinking behaviour paralleled that of studies involving either tropical natives or heat-acclimated participants . FurtherThough we focused primarily on examining hydration behaviour, the study also provided some insights into the physiological characteristics of SSG. The structure of the SSG performed in this study involved six players (3v3) on a full-court with both baskets in play. The duration of active gameplay was shorter in the SSG compared to traditional 5v5 basketball. Heart rate data showed that the players spent an average of 44.9% of on-court playing time in the high-intensity zone  . The aveThe findings of this study must be interpreted in light of some limitations. The study adopted a between-group study design with a small sample size. Future studies could use a larger sample size and/or adopt a within-group study design.Another methodological limitation was that the acceptance of artificial sweeteners was not assessed in the sensory questionnaire. Mouthfeel sensations, unlike the sensation of tastes , are “tactile, irritant and thermal” sensations . Most hiLastly, the present study only included male basketball players. Due to physiological sex differences , findings of this study could not be extended to female players. Future studies could consider examining sex differences in fluid preferences.This study highlights that fluid palatability, without added carbohydrates and/or electrolytes, does not induce any appreciable increase in fluid consumption compared to plain water in male collegiate basketball players during a practical game setting in the heat. In comparison to previous studies, it is plausible that electrolyte and carbohydrates concentrations might play a larger role than flavour in determining fluid consumption. Hence, practitioners could consider both fluid palatability and fluid composition in establishing a hydration plan for athletes. Our results also suggested the possibility that athletes’ on-field drinking behaviours may differ from their reported hedonic values. From an applied perspective, it may be meaningful to observe how athletes’ reported fluid preferences translate into their on-field hydration practices.


Nuclear imaging is a powerful non-invasive imaging technique that is rapidly developing in medical theranostics. Nuclear imaging requires radiolabeling isotopes for non-invasive imaging through the radioactive decay emission of the radionuclide. Nuclear imaging probes, commonly known as radiotracers, are radioisotope-labeled small molecules. Nanomaterials have shown potential as nuclear imaging probes for theranostic applications. By modifying the surface of nanomaterials, multifunctional radio-labeled nanomaterials can be obtained for in vivo biodistribution and targeting in initial animal imaging studies. Various surface modification strategies have been developed, and targeting moieties have been attached to the nanomaterials to render biocompatibility and enable specific targeting. Through integration of complementary imaging probes to a single nanoparticulate, multimodal molecular imaging can be performed as images with high sensitivity, resolution, and specificity. In this review, nanomaterial nuclear imaging probes including inorganic nanomaterials such as quantum dots (QDs), organic nanomaterials such as liposomes, and exosomes are summarized. These new developments in nanomaterials are expected to introduce a paradigm shift in nuclear imaging, thereby creating new opportunities for theranostic medical imaging tools. Molecular imaging is a non-invasive medical imaging technique capable of providing detailed images and information at molecular and cellular levels. Molecular imaging enables the visualization of cellular function through the different imaging modalities such as Positron Emission Tomography (PET) and Single-photon Emission Computed Tomography (SPECT). Imaging probes or biomarkers are used for tracking specific molecular pathways in particular targets in a living system ,2. The mBionanomaterials including organic and inorganic materials can be easily assimilated in living systems. These small-sized nanomaterials can penetrate into tiny capillaries and propagate across biological barriers, enabling detection of changes occurring at molecular levels. Combining these unique properties with being biocompatible has accelerated the application of nano-biomaterials for molecular imaging . Nanomat18F), carbon-11 (11C), gallium-68 (68Ga), iodine-124 (124I), and copper-64 (64Cu) for PET and technetium-99m (99mTc) for SPECT. 18Fluorinated 2-deoxy glucose (18F-FDG) is one of the most extensively used PET tracers. The design of 18F-FDG is for detecting the higher expression of glucose transporters in cancer cells Dalargin to poly(butylcyanoacrylate) nanoparticles injected systematically showed accumulations in the brain, indicating that the nanoparticle complex had crossed the BBB. The development of nanomaterials for specific targeting of the Central Nervous System (CNS) still remains extremely challenging due to the safety and efficacy concerns. However, there has been great advancement in the design of nanoparticle as carriers, drugs or probes to efficiently and safely propagate across the BBB. With more knowledge and better understanding of nanomaterials and their mechanisms, more success in the design of a nanomaterial probe may be achieved in targeting neurological diseases in the near future, creating more opportunities in therapeutics and diagnostic clinical applications.The limitations of crossing biological barriers such as the blood–brain barrier (BBB) and small capillaries can also be overcome using nanomaterials. These nanosized vessels are small enough to propagate across the barriers, delivering drugs to targeted sites . For exaBionanomaterials in particular have been widely investigated for theranostic applications due their natural ability to assimilate easily in body systems. These natural nanoparticles are equipped with unique properties such as biocompatibility, biodegradability, and stability in blood circulation, thereby overcoming some of the limitations that need to be considered in the design and development of a novel imaging probe . For exaTo improve their biocompatibility, targeting specificity, and easily controllable properties, nanomaterial-based nuclear imaging probes require necessary surface modifications such as coating and targeting moiety attachments, which include conjugation with peptides, antibodies, and many other targeting molecules . The modThe nanomaterial probe administered intravenously into the living system is assimilated either by the hepatobiliary system or by the renal system. To improve the biocompatibility, the nanoparticles are coated with hydrophilic polymers that include linear chain polymers of polyethylene glycol (PEG) and branched copolymers ,51. The Apart from PEG, several other coating polymers including polyvinyl pyrrollidone (PVP) , polyvinD. Dissociation constants determine the affinity with which the molecules can bind to each other. Melamide, succinimide, and other carbodiimide reactions are exploited to form covalent ester bond to link typically an alcohol group–carboxylic group to create an ester bond. Many such reactions are useful in molecular imaging for functionalizing the surface of the nanoparticles FBEM) for in vivo biodistribution and PET imaging studies Zr(oxinate)4 neutral complex. The radiometal complex was encapsulated into the exosomes and ensured that the surfaces of the exosome were not affected. The lipophilic complex [89Zr]Zr(oxinate)4 was able to pass through the lipid bilayer of the vesicles where 89Zr dissociated from the oxine ligands; dissociated free 89Zr then bound to intravesicular metal-chelating ligands, such as proteins and nucleic acids within the exosomes. Biodistribution studies using exosomes derived from pancreatic cancer cells (PANC1) via PET-CT displayed 89Zr-PANC1 exosomes accumulated in the liver, spleen, bladder, lymph nodes, and brain within 1 h of intravenous (iv.) injection [Intraluminal labeling of radionuclides to exosomes was demonstrated through the use of a metastable lipophilic [njection . As exosWith the development of nuclear imaging probes using nanomaterials, pathological processes at molecular levels can be revealed that could potentially help in early detection and interventions of various diseases. Enhancement of nanomaterial targeting capabilities as imaging probes has been explored through various approaches such as surface modifications and targeting moieties attachment. In this review, we summarized developments of nanomaterials as nuclear imaging probes through various modifications. The applications of nanomaterials have been extensively exploited for biocompatibility, in vitro stability, and in vivo biodistribution and animal imaging. Despite promising results from in vivo studies, there are still challenges that need to be overcome before nanomaterials can be applied as diagnostic and/or therapeutic nuclear medicine and imaging tools in clinical applications. For example, the reproducibility of various nanomaterials needs to be systematically determined, and the synthetic methods need to be standardized to ensure their biocompatibility so that theoretical modelling of nanomaterial integration can be guided for cell tracking and cell internalization. The review provides some future research directions to those with expertise in radiochemistry, nuclear imaging engineering, and other bio-imaging research. It is envisaged that the research thrust area combining these nanomaterials including exosomes can lead to a new generation of nuclear imaging probes and introducing a paradigm shift in theranostics.


Analyzing Twitter posts enables rapid access to how issues and experiences are socially shared and constructed among communities of health service users and providers, in ways that traditional qualitative methods may not.To enrich the understanding of mental health crisis care in the United Kingdom, this study explores views on crisis resolution teams (CRTs) expressed on Twitter. We aim to identify the similarities and differences among views expressed on Twitter compared with interviews and focus groups.We used Twitter’s advanced search function to retrieve public tweets on CRTs. A thematic analysis was conducted on 500 randomly selected tweets. The principles of refutational synthesis were applied to compare themes with those identified in a multicenter qualitative interview study.#CrisisTeamFail, where posts were principally related to poor quality of care and access, particularly for people given a personality disorder diagnosis. Posts about CRTs giving unhelpful self-management advice were common, as were tweets about resource strains on mental health services. This was not identified in the research interviews. Although each source yielded unique themes, there were some overlaps with themes identified via interviews and focus groups, including the importance of rapid access to care. Views expressed on Twitter were generally more critical than those obtained via face-to-face methods.The most popular hashtag identified was Traditional qualitative studies may underrepresent the views of more critical stakeholders by collecting data from participants accessed via mental health services. Research on social media content can complement traditional or face-to-face methods and ensure that a broad spectrum of viewpoints can inform service development and policy. Twitter has emerged as a prominent social media platform that may be a valuable data source for researchers wanting to access stakeholders’ views on many topics, including mental health services . Resultsreactive way, conveying in-the-moment feelings and experiences  don’t know what to do with them, which is fine, but neither do we.CORE CRT staffpersonality disorder.In summary, although both data sets show a mixed landscape of experiences, Twitter users often reported more difficulty accessing CRT care. This was particularly the case for Twitter users who were posting about Both service users and carers in the CORE study reported mixed experiences of the quality of care received from CRTs. In the CORE interviews, help with practical issues, emotional support, and relationship building were all described as important aspects of good quality care. Service users valued times when they received emotional support from staff who listened to them and came across as caring. There was a wish among some stakeholders for a more holistic approach to care, for example, more support with social issues. This was not observed in the Twitter sample:They did listen to me. They did understand my predicament. They acknowledged my dilemma about taking a medication.CORE service userCrisis team advising me to take a walk in the park is probably the worst thing to say considering I make self-harm plans in the parkTwitter userImportantly, a significant aspect of care quality that was frequently discussed by service users on Twitter was receiving unhelpful self-management advice from the CRT. This was unique to Twitter: in the CORE data set, stakeholders did not report experiences of feeling dismissed by distraction or self-care advice.Both data sets identified staff characteristics as a determinant of good quality treatment, with consensus that there is a wide variety of staff expertise within CRTs. As CRT staff work shifts, the extent to which consistent therapeutic relationships are established varies greatly, and continuity of care is a challenge. This was reflected in both data sets, where continuity of staffing was valued and seen as important in CRT care. Personal staff characteristics, such as empathy and compassion, were consistently described in both forms of data as critical ingredients in good CRT support:He’s had some good, really good staff come to see him, and he’s had some damn awful, pretty diabolical people come to see him, as well.CORE carerShocked by reading the hashtag #crisisteamfail, I have experienced this but I also think some CRT staff are greatTwitter userIn our sample, we found that people posted on Twitter about a strain on mental health services due to high demands and a lack of resources. This theme was unique to the Twitter data set and was not discussed in the CORE interviews and focus groups with service users, despite the broad semistructured nature of the interview questions. Some Twitter users directed tweets toward the Twitter accounts of the NHS and UK government. This theme reflects the political nature of Twitter as a social forum in comparison with interviews and focus groups that take place in a private space. This theme was distinct from the theme “the role of CRTs in the care system” in the CORE data set, which described the role of CRTs as gatekeepers for hospital admissions:NHS mental health crisis services are being stretched to breaking point.Twitter userPolice are helping people who are self-harming cos the conservatives slashed funding for CRTs.Twitter userpersonality disorder diagnoses discussed their diagnosis as a barrier to accessing CRT care. More generally negative sentiments were expressed on Twitter compared with traditional face-to-face data collection methods. Therefore, it seems likely that more negative experiences of CRT service use and implementation may be missed by relying solely on face-to-face data collection methods.Our findings suggest that the analysis of Twitter data can complement traditional qualitative research methods and expand our understanding of stakeholder views of mental health crisis care delivered by CRTs. Many of the same things feature in what service users and family caregivers describe as valuable components of good quality CRT care in both fora, particularly staff who listen and provide warmth and empathy. We accessed additional insights into the experiences of CRTs from Twitter. Twitter users posted that self-management strategies suggested by CRT staff were frequently experienced as unhelpful and expressed concern about the strain on mental health services. Twitter users with spaces for reflection. This is encouraged in numerous ways: at the time of data collection , in the open nature of questioning, and in the way in which interviews are introduced as research where there are no right or wrong answers [heat of the moment [interview room.Twitter differs from traditional qualitative methods in several respects. Interviews and focus groups are often set up as  answers . The inte moment ,52. TwitTwittersphere. For example, interactions about unhelpful self-management advice appear to have turned into a running joke in this Twitter community as part of the hashtag #crisisteamfail. This is a clear example of how views are constructed and reinforced in the social sphere of Twitter in a way that is very different from how views are expressed in individual face-to-face methods. Analyzing Twitter posts can therefore allow access to how issues and experiences are shared among socially mediated communities of health service users [Twitter is set up as a social media forum, encouraging interactions between users ,53,54. Tce users ,55.personality disorders in the CORE interviews had received a service from the CRT, so the sample was unlikely to include people who felt that they had been denied care. The Twitter analysis may therefore allow research to access a broader range of voices compared with face-to-face qualitative methods alone.The participant recruitment processes for interviews and focus groups in the CORE study likely introduced some selection bias, as potential participants were identified via CRTs. This necessarily excludes people in mental health crises who could not or did not want to engage with CRTs. As the CRT staff contributed to identifying service users and their family members, they may have been more likely to identify service users who had engaged positively with CRT care . For example, participants diagnosed with Individuals who are active on Twitter may differ from those who engage in traditional qualitative research and may differ from CRT service users as a whole. A significant proportion of mental health service users (and nonusers) are distrustful of services or may have had negative previous contacts with services, sometimes involving coercion. These individuals may be more inclined to engage in Twitter than in traditional research projects. In addition, given its broad social reach, people who use Twitter may be more likely to engage as advocates and activists, bringing this distinctive point of view to the table. Twitter can be viewed as a naturalistic public setting in which service users can have their voices heard , gaininghashtags, which link tweets together. By conducting the search directly from Twitter, we avoided bias that would have been introduced if we used software such as NCapture [This is the first study to compare views expressed on Twitter with data collected from interviews and focus groups in the field of mental health research. A broad range of search terms were used, both with and without NCapture . The benNCapture  are equaThis study has some limitations. The search strategy meant that the demographics of tweeters could not be retrieved. Tweets were accessed through Twitter’s advanced search function, which uses Twitter’s application programming interface. This means that the search output is determined in part by Twitter’s programming. Nevertheless, research suggests that the application programming interface method still retrieves a large proportion of tweets available in the public domain . The seaThe refutational synthesis found that there are some overall congruent messages about what is considered important in CRT practice; however, there were differing views about how far this is being achieved and some themes emerged from only one set of analyses. Our confidence in CORE study findings  regardinOur analysis suggests that CRTs need to develop new approaches when supporting individuals with self-management advice and that CRT staff should avoid giving general self-care advice, such as going for walks. This is likely to apply to other clinical contexts in mental health care. Instead of self-care advice, self-management strategies should be tailored toward relapse prevention and recovery goals . For exapersonality disorder, as our Twitter analysis showed that this group often felt dismissed, neglected, and stigmatized by services. Further explorations are desirable to examine the potential of Twitter and other social media platforms in mental health research. We hope that there will be developments in evidence-based best practice for conducting qualitative research using Twitter data and for combining social media analysis with standard interviews.Researchers may find it beneficial to engage proactively with Twitter to gain a broad range of perspectives. This is particularly the case when research questions seek to include those who have difficulty engaging or have disengaged from services or where studies have difficulty recruiting participants for lengthy interviews and focus groups. Future research using Twitter data may use natural language processing techniques to examine the broad content of Twitter posts. It would also be interesting to track trends and changes over time. A limitation of this study was that we could not conduct any causal analysis, and future research may explicitly ask CRT stakeholders to reflect on and explain differences between themes in the Twitter and interview data sets. The themes identified in this study demonstrate that more research is needed on how best to optimize crisis care for people who have been diagnosed with a Twitter users provided unique perspectives on CRT implementation, including the importance of the type of advice offered to help manage crises and the perceived limitations of CRT services. Research on social media content adds complexity to our understanding of phenomena and can complement traditional or face-to-face research methods in health care research by allowing the voices of people who may be more critical of services to be heard.


Streptococcus pneumoniae (SPN), Haemophilus influenzae (HI), Haemophilus parainfluenzae (HP), methicillin-susceptible Staphylococcus aureus (MSSA), Escherichia coli (EC), Klebsiella pneumoniae (KPN), Pseudomonas aeruginosa (PSA), Chlamydia pneumoniae, Mycoplasma pneumoniae and L. pneumophila. In this study, the in vitro susceptibilities of DLX and comparator quinolones were determined for clinical isolates from CAP and CA-lower respiratory tract infections (LRTIs).Delafloxacin (DLX) is a broad-spectrum fluoroquinolone antibacterial approved in the US for the treatment of community-acquired bacterial pneumonia (CABP). DLX is indicated to treat CABP caused by CAP and CA-LRTI isolates were consecutively collected at 67 US medical centers participating in the SENTRY Surveillance Program during 2014-2020. Sites submitted 1 isolate per patient per infection episode. Isolate identification was determined at each site and confirmed using standard biochemical or molecular methods at JMI Laboratories. Susceptibility testing was performed according to CLSI broth microdilution methodology. CLSI (2021) interpretive criteria were applied, FDA criteria were used for DLX. The susceptibility results for DLX, levofloxacin (LEV), moxifloxacin (MOX), and ciprofloxacin (CIP) for the indicated species are shown in the table. As MOX does not have CLSI breakpoints for EC, KPN, or PSA, CIP was tested for those species instead. DLX had the highest percent susceptibility against MSSA (91.8%). SPN and HI were >97% susceptible, and HP was >91% for all 3 drugs. KPN susceptibility ranged from 86.4% for LEV to 76.9% for DLX. Susceptibilities for EC and PSA were similar for the 3 drugs, EC varied from 59.8% for LEV to 57.0% for DLX, and PSA varied from 71.6% for CIP to 64.0% to LEV. in vitro results suggest that DLX may be a useful therapeutic option for CABP caused by Gram-positive, Gram-negative and fastidious pathogens.DLX had good activity against recent CAP and CA-LRTI isolates from US hospitals. DLX had the highest susceptibility of the quinolones tested against MSSA. Quinolone-resistant SPN and HI were uncommon. These Dee Shortridge, PhD, AbbVie (formerly Allergan) (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Shionogi (Research Grant or Support) Jennifer M. Streit, BS, GlaxoSmithKline, LLC (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Michael D. Huband, BS, AbbVie (formerly Allergan) (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support) Mariana Castanheira, PhD, AbbVie (formerly Allergan) (Research Grant or Support)Bravos Biosciences (Research Grant or Support)Cidara Therapeutics, Inc. (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Qpex Biopharma (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Mariana Castanheira, PhD, Affinity Biosensors  Involved: Self): Research Grant or Support; Allergan  Involved: Self): Research Grant or Support; Amicrobe, Inc  Involved: Self): Research Grant or Support; Amplyx Pharma  Involved: Self): Research Grant or Support; Artugen Therapeutics USA, Inc.  Involved: Self): Research Grant or Support; Astellas  Involved: Self): Research Grant or Support; Basilea  Involved: Self): Research Grant or Support; Beth Israel Deaconess Medical Center  Involved: Self): Research Grant or Support; BIDMC  Involved: Self): Research Grant or Support; bioMerieux Inc.  Involved: Self): Research Grant or Support; BioVersys Ag  Involved: Self): Research Grant or Support; Bugworks  Involved: Self): Research Grant or Support; Cidara  Involved: Self): Research Grant or Support; Cipla  Involved: Self): Research Grant or Support; Contrafect  Involved: Self): Research Grant or Support; Cormedix  Involved: Self): Research Grant or Support; Crestone, Inc.  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Taking into account the possibility of myelin-associated proteins having a role in brain tumour development, the study aimed to evaluate the diagnostic usefulness of myelin-associated proteins  released into extracellular space in patients with brain tumours.n = 49) and non-tumoural subjects (n = 24) was measured in cerebrospinal fluid (CSF) and serum by means of ELISA. Immunohistochemistry for IDH1-R132H was done on 5-μm thick formalin-fixed, paraffin-embedded tumour sections with the use of an antibody specific for the mutant IDH1-R132H protein.Protein concentration in primary brain tumour (IDH1 wild-type astrocytic brain tumour patients compared to IDH1 mutant patients.The receiver operator characteristic curve analysis showed that CSF Nogo-A and serum MAG were useful in differentiating patients with primary brain tumour from non-tumoural individuals. This was also true in the case of the separate analysis of the astrocytic tumour versus non-tumoural groups and the meningeal tumour versus non-tumoural groups. Neither Nogo-A nor MAG or OMgp concentrations were significantly different, in serum or CSF, between Myelin-associated proteins may be circulating brain tumour biomarkers.Nogo-A and MAG proteins seem to be the most useful in brain tumour diagnosis.Decreased CSF Nogo-A concentration is an adverse prognostic factor for patients’ survival.Our results indicated the potential usefulness of CSF Nogo-A and serum MAG evaluation as circulating biomarkers of primary brain tumours. Because blood is relatively easy to obtain, future research should be conducted to explicitly indicate the value of serum MAG concentration evaluation as a brain tumour biomarker. Glioma is usually detected too late, when it has infiltrated the brain to a major extent, thus preventing early intervention. The discovery of a circulating biomarker for these tumours would be of great importance .Neurite growth isoform A of reticulon-4 (Nogo-A), myelin-associated glycoprotein (MAG), and oligodendrocyte myelin glycoprotein (OMgp) are all myelin-derived proteins. Nogo-A expression is found in neurons and oligodendrocytes of the central nervous system in adults . MAG is After the injury, such as those occurring during glioma invasion, Nogo-A, MAG, and OMgp are released from the cell membrane to the extracellular space. Their growth-inhibitory signal is transmitted via the cell surface receptor complex including Nogo Receptor 1 (NgR1), Lingo-1, p75NTR, and TROY. The recently paired immunoglobulin receptor B protein (PirB) has also been identified as an additional transmembrane receptor with a high affinity to bind myelin-associated proteins .in vitro [Although the involvement of Nogo-A, MAG, and OMgp in the inhibition of central nervous system regeneration has been widely studied , the datin vitro . The rolIDH) glioma mutants showed a better prognosis compared to IDH-wildtype glioma patients, in the astrocytic brain tumour subgroup we also analyzed Nogo-A, MAG, and OMgp levels depending on the IDH1 gene mutation. Finally, we examined the relationship between the tested proteins and patient diagnosis and survival.Taking into account the possibility of myelin-associated proteins having a role in brain tumour development, the study aimed to evaluate whether CSF and serum MAG and OMgp concentrations are different between astrocytic and meningeal tumour patients compared to non-tumoural individuals. In the next step, we compared the diagnostic utility of evaluating the three myelin-associated proteins of interest  in patients with primary brain tumour. Because isocitrate dehydrogenase (2.2.1.N = 33) and patients with tumours of the meninges (N = 16). For individual patient characteristics see Supplementary Materials  with previously untreated primary brain tumours: patients with astrocytic brain tumours  within 0.5 h after collection. Obtained serum and CSF supernatant samples were aliquoted and stored at –75 °C until further analysis.Patients’ blood was collected in 2.7-mL test tubes without anticoagulant . CSF and blood samples were centrifuged . Informed consent was obtained from all subjects involved in the study.2.3.To determine whether myelin-associated protein concentration changed in individuals with brain tumours, we first evaluated protein levels in CSF and serum. Nogo-A concentration was analyzed in 49 patients with brain tumour and 24 non-tumoural individuals. MAG and OMgp concentrations were analyzed in 30 patients with brain tumour and 10 non-tumoural subjects.Nogo-A, MAG, and OMgp concentrations were analyzed by means of the enzyme-linked immunosorbent assay (ELISA) method in compliance with the manufacturer’s instructions. CSF and serum samples were not diluted before analysis.Nogo-A concentration was measured using a Human RTN4 ELISA kit  from Wuhan Fine Biological Technology Co., Ltd. The detection range for the kit is between 78.125 and 5000 pg/mL, the minimum detectable dose (sensitivity) of the assay is <46.875 pg/mL. The sensitivity of this assay was defined as the lowest protein concentration that could be differentiated from 0.00 pg/mL. It was determined by the mean O.D. value of 20 replicates of the zero standard added to their three standard deviations (SD). Values <46.875 ng/mL were considered as 0.00 ng/mL.MAG concentration was measured using the Human MAG ELISA kit  from Biorbyt Ltd., Cambridge, England. The detection range for the kit is between 0.625 and 40 ng/mL, the sensitivity of the assay is 0.156 ng/mL. The sensitivity of this assay was defined as the lowest protein concentration that could be differentiated from 0.00 pg/mL. It was determined by the mean O.D. value of 20 replicates of the zero standard added to their three standard deviations (SD). Values <0.156 ng/mL were considered as 0.00 ng/mL.OMgp concentration was measured using the Human OMG ELISA kit  from Biorbyt Ltd., Cambridge, England. The detection range for the kit is between 0.156 and 10 ng/mL, the sensitivity of the assay is 0.039 ng/mL. The sensitivity of this assay was defined as the lowest protein concentration that could be differentiated from 0.00 pg/mL. It was determined by the mean O.D. value of 20 replicates of the zero standard added to their three standard deviations (SD). Values <0.039 ng/mL were considered as 0.00 ng/mL.2.4.IDH1 mutation, representing above 90% of the IDH mutations existing in gliomas [ gliomas , is deteCombined cytoplasmic and nuclear staining was interpreted as immunopositive. Three-tiered semiquantitative system results were as follows: if no tumour cell was immunopositive – it was reported as negative; if there was an admixture of immunopositive and immunonegative tumour cells or areas of immunopositive and immunonegative tumour cells were adjacent to each other – it was reported as partly/focal positivity; if all tumour cells were immunopositive – it was reported as complete (diffuse) positivity.2.5.p < .05.The obtained results were statistically analyzed with the use of the STATISTICA 12.0 PL software , STATA 12.1 (StataCorp LP), and GraphPad Prism 5.0 . The concentration of the tested parameters did not follow a normal distribution (Shapiro–Wilk test), thus nonparametric statistical analysis was employed. The Mann–Whitney test was used to compare two independent samples, and the ANOVA rank Kruskal–Wallis test was used for the comparison of three samples. Values for measured variables are stated as median with 25th and 75th percentiles . Receiver operator characteristic (ROC) curve was generated to calculate the area under the ROC curve (AUC) ,18. The 3.3.1.p < .001). ANOVA rank Kruskal–Wallis indicated that both, astrocytic and meningeal tumour subgroups had statistically lower CSF Nogo-A concentrations compared to non-tumoural individuals   .p = .508). The ANOVA rank Kruskal–Wallis test did not show a statistical difference for CSF MAG concentrations   .H = 5.86; df = 2; p = .053)  .Altogether the aforementioned data demonstrate that from all the tested proteins only CSF Nogo-A significantly differentiated patients with brain tumour from non-tumoural individuals. Thus for further diagnostic utility, analysis of only CSF Nogo-A was pursued.3.2.p = .004). ANOVA rank Kruskal–Wallis showed a statistically significant difference between patients with astrocytic brain tumour and non-tumoural individuals  as well as between patients with meningeal tumour and non-tumoural individuals (p = .008)  .p = .031). The ANOVA rank Kruskal–Wallis test showed a statistically significant difference between patients with meningeal brain tumour and non-tumoural individuals   .The aformentioned data demonstrate that MAG and OMgp could both be potentially useful biomarkers in the serum. However, for further diagnostic utility analysis, we decided to pursue only serum MAG. The reason for our decision is presented in the Discussion section.3.3.p < .001). On the contrary, in the non-tumoural group median CSF MAG concentration was not statistically different compared to the serum value . These results indicate differing serum MAG expressions in patients with brain tumour and non-tumoural subjects.To investigate whether MAG expression differs between the central and peripheral nervous system, we compared CSF to serum values. Patients with brain tumour had significantly higher median CSF MAG concentrations compared to serum MAG values  glioma mutants showed a better prognosis compared to IDH-wildtype glioma patients at different WHO grades [IDH1 gene mutation in the astrocytic brain tumour subgroup. Neither Nogo-A nor MAG or OMgp concentrations were significantly different, in serum or CSF, between patients with IDH1 wild-type astrocytic brain tumour compared to patients with IDH1 mutant astrocytic brain tumour (data not shown), which may indirectly indicate that the tested molecules could not be recognized as prognostic biomarkers of brain tumours.Because the available literature indicates that isocitrate dehydrogenase  with an increase in K + concentration by 1 mg/dL, the chances of brain tumour presence increase by 3.71-fold; (5) with an increase in glucose concentration of 10 mg/dL, the chances of brain tumour presence increase by 25.37%; and (6) with an increase in urea concentration by 1 mg/dL, the chances of brain tumour presence increase by 9.00% , potassium (K+), glucose, and urea concentrations. We showed that: (1) the chances of developing a brain tumour in men are almost four times higher than in women; (2) with an increase in CSF Nogo-A by 100 pg/mL, the chances of a brain tumour decrease by 6.11%; (3) with an increase in WBC by 1 × 10by 9.00% .In the model of multivariate logistic regression analysis, predictor variables influencing brain tumour diagnosis included: CSF Nogo-A, K+, urea, and creatinine concentrations. We showed that: (1) with an increase in CSF Nogo-A by 100 pg/mL, the chances of brain tumour presence decrease by 15.03%; (2) with an increase in K + concentration by 1 mg/dL, the chances of brain tumour presence increase by more than 17-fold; (3) with an increase in urea concentrations by 1 mg/dL, the chances of brain tumour presence increase by 23.00%; (4) with an increase in creatinine concentration by 0.01 mg/dL, the chances of developing a brain tumour decrease by 6.76% .3.6.The diagnostic utility analysis found that both CSF Nogo-A and serum MAG were useful in differentiating patients with primary brain tumour from non-tumoural individuals. This was also true in differentiating individual brain tumour subgroups, patients with astrocytic brain tumour, or patients with meningeal brain tumour, from non-tumoural subjects. Our findings present CSF Nogo-A and serum MAG as being potentially useful circulating biomarkers of primary brain tumours .3.7.p = .102). Interestingly, when separately analyzing males and females, we found that the probability of survival was significantly greater for females with higher CSF Nogo-A concentrations .N = 11) and non-tumoural individuals (N = 7). This is interesting because according to the literature OMgp is exclusively expressed only within the central nervous system [An unexpected result was the finding of some serum OMgp concentrations in both primary brain tumours  from non-tumoural individuals. The diagnostic utility analysis showed that the highest AUC (0.909), Youden Index (0.65), diagnostic specificity (100%), and positive predictive value (PPV) (100%) were found for CSF Nogo-A. Serum MAG displayed less worth in differentiating the total group of patients with primary brain tumour from non-tumoural individuals . In other words, the evaluation of CSF Nogo-A concentrations was found to be the most suitable for primary brain tumour diagnosis, as the specificity of the test reached 100%. It is well-established that the higher the specificity, the smaller the number of false-positive results ,26.A deeper analysis of the NgR1 ligands found that CSF Nogo-A evaluation had lower diagnostic specificity (83%) compared to serum MAG (90%) when differentiating patients with astrocytic brain tumour from non-tumoural individuals. However, serum MAG had lower AUC (0.770), but higher PPV (93%) compared to CSF Nogo-A . In the differential diagnosis of patients with meningeal brain tumour from non-tumoural individuals, CSF Nogo-A evaluation was the most appropriate test with AUC = 0.987 and a specificity of 100%. However, it should be noted that both PPV and negative predictive value (NPV) are likely to be interpreted depending on the prevalence of the disease ,27.The diagnostic utility analysis indicated the usefulness of CSF Nogo-A and serum MAG concentration evaluation to confirm primary brain tumour diagnosis. However, it is worth mentioning that in our case–control study the brain tumour group and the non-tumoural group were relatively small, thus estimated AUCs could be biased due to relatively few points on the curve. Other factors influencing the diagnostic accuracy analysis may be related to sampling bias, prevalence variability due to inappropriate study design, clinical patient variability, subgroup differences, or even reader expectations . UndoubtKaplan–Meyer curve analysis demonstrated that the probability of survival was significantly greater for females with higher CSF Nogo-A concentrations. This could be related to the fact that our previous study presented, utilizing multiple linear regression analysis, that the mean CSF Nogo-A concentration was 1.9 times higher for women in comparison to men . NeverthIt was also interesting for us to analyze how biochemical parameters routinely done for all patients with brain tumour may predict the probability of brain tumour diagnosis. Interestingly, multivariate logistic regression analysis showed that predictor variables influencing brain tumour diagnosis included: CSF Nogo-A, K+, urea, and creatinine concentration. There is no similar analysis in the available literature, which makes it impossible to discuss the obtained results. Undoubtedly, the fact that routinely evaluated biochemical parameters can predict the probability of brain tumour diagnosis is worth exploring in further studies.One limitation of the study could be the sensitivity of the applied ELISA tests for Nogo-A, MAG, and OMgp concentration evaluation. The standard curve points of MAG and OMgp ELISA kits were expressed in ng/mL. On the contrary, for Nogo-A the standard curve points were expressed in pg/mL. These differences in the sensitivity of the ELISA kits may explain why some MAG and OMgp results were below the detection range. Another study limitation could be the specificity of the applied ELISA kits. Thus, we asked the ELISA kit suppliers for the anti-Nogo-A, anti-MAG, and anti-OMgp antibodies specificity. In the case of the Nogo-A test, the ELISA vendor used UniProtKB: Q9NQC (1-200aa) antibody, which is only human-specific. In the case of the MAG and OMgp tests, we have obtained information that the used kits did not reveal any cross-reactivity with myelin basic protein (MBP), myelin protein zero, and myelin protein two. The possible cross-reactivity of the OMgp ELISA kit with GPIbαβ was not tested.It must be also acknowledged, that our “control group” did not include entirely healthy people, as they had diagnosed unruptured brain aneurysms. Nevertheless, despite this limitation, we still were able to demonstrate statistically significant differences. Moreover, there was a sex disparity in non-tumoural subjects, which was dictated by fact that female sex is a risk factor of brain aneurysms. Finally, to make a quantitative tumour marker clinically applicable for cancer diagnosis, it is necessary to establish reference values in the control group or evaluate a marker in a follow-up with a single patient . None of5.We demonstrated significantly decreased CSF Nogo-A concentration in patients with primary brain tumour  compared to non-tumoural individuals. We also found that serum MAG concentration is significantly decreased in these patients compared to control subjects. The obtained results indicated CSF Nogo-A and serum MAG evaluation as potential circulating diagnostic markers of primary brain tumours. We believe that the study we conducted provides cognitive knowledge about quantitative myelin-associated protein evaluation in patients with primary brain tumour.Click here for additional data file.


Anand TN, Joseph LM, Geetha AV, Prabhakaran D, Jeemon P. Task sharing with non-physician health-care workers for management of blood pressure in low-income and middle-income countries: a systematic review and meta-analysis. Lancet Glob Health 7: e761–712019; —The acknowledgments section of this article should have stated that “This work was supported by the Wellcome Trust/DBT India Alliance Fellowship (grant number IA/CPHI/14/1/501497) awarded to Panniyammakal Jeemon”. This change has been made to the Article as of Aug 28, 2019.


Intra-scrotal swellings are commonly encountered all over the globe. Common causes include hydroceles, hernias, lipoma, cysts, etc. Rarely manifested as scrotal swelling is paratesticular swelling arising outside the testis. Clinically, they may mimic a tumour arising from testis either benign or malignant with grave consequences. Diagnosis hence is of paramount importance. It has to be ruled out as a diagnosis of exclusion. Knowledge of such swelling those other than the ones originating from the testis is thus crucial in order to have a better patient outcome in the absence of any mandated published guideline. Infrequent in incidence, para/extra-testicular neoplasms are clinically noteworthy affecting all males. These tumours are asymptomatic, benign with/without harmful consequences manifesting as a small swelling in the inguinoscrotal region . The gloA 40-year-old male labourer had painless, insidious, slow-growing swelling in the left scrotum for one year. Significant complaints of mild pain in the left scrotum while walking led to the detection of this swelling. Physical examination revealed non-tender, firm swelling felt just above the upper pole of the testis along the spermatic cord.USG scrotum unveiled isoechoic, homogeneous, avascular soft-tissue mass of 3.2cm x 3cm in left hemiscrotum, along spermatic-cord discrete from left testis suggestive of a probable benign epididymal adenomatoid tumour Figure . As the Intra-operatively, 3cm oval mass was discovered at the upper pole of the testis arising from the head of the epididymis although lying separately from the testis. Hence an en bloc excision of the mass was accomplished without damaging structures followed by a frozen section which confirmed the diagnosis of an adenomatoid tumour. The macroscopic cut section revealed a solid, homogenous, smooth-surfaced, whitish-yellowish lesion. Histopathology confirmed the diagnosis of adenomatoid tumour of epididymis with the presence of tubular epithelium and stromal cuboidal cells. Immunohistochemical positivity for adenomatoid-specific markers  was further added to the confirmation of diagnosis Figure . The patGlobally paratesticular tumours are rarely encountered with an overall incidence of less than 5%. Data regarding the incidence of paratesticular tumours in Toto or epididymal tumours per se in India are also missing due to a lack of conducted studies and knowledge about these cases. Lipoma of the spermatic cord is a popular benign paratesticular tumour and in the epididymis, the most common is adenomatoid tumour followed by leiomyoma, fibroma, haemangioma, neurofibroma and papillary cystadenoma. Such benign tumours also mimic an extra-testis (polyorchidism) ,2. In 20The adenomatoid tumour is commonly found benign tumour of epididymis with an incidence of 73% followed by papillary cystadenoma 11%), leiomyoma (9%) . Embryol1%, leiomThese tumours are not age influenced but are found in ages between 18 and 80 years ,6,7. PatUltrasonography is the initial investigation of choice as it is easily available, cost-effective, and diagnostic with outrageous sensitivity and specificity. It differentiates between a benign or malignant lesion, demarcates boundaries and describes echogenicity, vascularity, invasive behaviour and adjacent structures. If USG reveals a well bordered, solitary, homogenous, non-invasive lesion further use of CECT or MRI can be limited. If USG findings are unclear or doubtful, further radiology can be performed using CT or MRI and sending tumour markers for testicular malignancy for evaluation. FNAC can be performed but in over case was avoided since USG diagnosed a benign lesion which was then planned for excision ,2,4,7.Gross examination reveals a solitary, smooth, extra-testicular white-yellow nodule-like lesion. Microscopy reveals patterns of tubules, cords and nests with cuboidal epithelium and vacuolated/eosinophilic cytoplasm along with fibrous hyalinised stroma. Pathological differentials incorporate metastatic carcinoma, malignant mesothelioma, histiocytoid haemangioma and carcinoma of the rete testis. Here comes the use of Immunohistochemical analysis which helps in the final confirmation of diagnosis by differentiating benign from malignant and mesothelial from non-mesothelial. The mesothelial origin of adenomatoid is confirmed by the existence of Calretinin which is a calcium-binding protein of the S-100 family. It shows an exorbitant amount of sensitivity for the detection of mesothelial cells. Other marker includes HMB1, CK 5/6, vimentin, CK EMA and WT1 ,7.Treatment of choice is surgical which includes confirmative diagnosis by frozen section followed by complete resection of the tumour by scrotal approach without man-handling of adjacent structures. If the frozen section diagnoses a benign tumour, no further exploration is indicated as there is no evidence of recurrence reported in the literature of adenomatoid tumour to date -3,7.After acknowledging current literature we found sparse case reports of epididymal adenomatoid tumour in the last five years, specifically from India. Considering the rarity and importance of diagnosis to rule out malignancy, we are presenting this case with a motif of creating awareness regarding such benign tumours, which should be addressed during primary surveillance. The creation of appropriate guidelines for pre-operative, intra-operative and post-operative management of such cases is a must for correct diagnosis and overall prognosis of the patient.


Tebipenem pivoxil hydrobromide (TBP-PI-HBr) is an oral prodrug that is converted to tebipenem (TBP), the active moiety. TBP is a carbapenem with activity against multidrug-resistant Gram-negative pathogens, including extended-spectrum-β-lactamase-producing Enterobacterales and is being developed for treating complicated urinary tract infections (cUTI) and acute pyelonephritis (AP). Data from three Phase 1 studies and one Phase 3 study in patients with cUTI/AP were used to develop a population pharmacokinetic (PPK) model for TBP and identify covariates that described the variability in TBP pharmacokinetics (PK).The PPK model was developed using TBP plasma and urine concentration-time data from the above-described Phase 1 and 3 studies. TBPPI-HBr doses, which ranged from 100 to 900 mg, were administered as single or multiple doses every 8 hours. After development of the structural model, stepwise forward and backward selection procedures were used to identify significant covariate relationships. The robustness of the final PPK model was assessed using a prediction-corrected visual predictive check (PC-VPC).The final dataset included 3448 plasma concentrations from 99 Phase 1 subjects and 647 Phase 3 patients and urine concentrations from 128 Phase 1 subjects. A two-compartment model with linear, first-order elimination and transit compartments to describe the rate of drug absorption after oral administration of TBP-PI-HBr best described TBP PK. The most clinically significant covariate effect, which would warrant dose adjustment, was the relationship between apparent oral clearance and creatinine clearance. In contrast, age, body size, sex, and fed status each had a minimal impact on TBP exposure. The PC-VPC showed good agreement between median simulated plasma concentrations based on the final PPK model and the median observed plasma concentrations for the pooled dataset .Figure 1. Prediction-corrected visual predictive check plot for the final population PK model using the pooled analysis datasetA robust description of TBP plasma PK in subjects and patients with cUTI/AP was achieved, such that derived measures of TBP plasma exposure are expected to be both accurate and precise. The population PK model was considered appropriate for model-based simulations and the assessment of PK-PD relationships for TBP.Harish Ganesan, M.S., 3-V Biosciences (Grant/Research Support)Achogen (Grant/Research Support)Amplyx Pharmaceuticals, Inc. (Grant/Research Support)Arixa Pharmaceuticals (Grant/Research Support)Arsanis Inc. (Grant/Research Support)B. Braun Medical Inc. (Grant/Research Support)Basilea Pharmaceutica (Grant/Research Support)BLC USA (Grant/Research Support)Boston Pharmaceuticals (Grant/Research Support)Bravos Biosciences, LLC (Grant/Research Support)Cidara Therapeutics Inc. (Grant/Research Support)Cipla, USA (Grant/Research Support)Corcept Therapeutics (Grant/Research Support)Cumberland Pharmaceuticals (Grant/Research Support)Debiopharm International SA (Grant/Research Support)Discuva Limited (Grant/Research Support)Emerald Lake Technologies (Grant/Research Support)Enhanced Pharmacodynamics (Grant/Research Support)Entasis Therapeutics (Grant/Research Support)E-Scape Bio (Grant/Research Support)Genentech (Grant/Research Support)Geom Therapeutics, Inc. (Grant/Research Support)GlaxoSmithKline (Grant/Research Support)Hoffmann-La Roche (Grant/Research Support)Horizon Orphan LLC (Grant/Research Support)ICPD Biosciences, LLC (Grant/Research Support)Indalo Therapeutics (Grant/Research Support)Insmed Inc. (Grant/Research Support)Institute for Clinical Pharmacodynamics (Employee)Iterum (Grant/Research Support)KBP Biosciences USA (Grant/Research Support)Kyoto Biopharma, Inc. (Grant/Research Support)Matinas (Grant/Research Support)Meiji Seika Pharma Co., Ltd. (Grant/Research Support)Melinta Therapeutics (Grant/Research Support)Menarini Ricerche S.p.A. (Grant/Research Support)Merck & Co., Inc (Grant/Research Support)Mutabilis (Grant/Research Support)Nabriva Therapeutics AG (Grant/Research Support)Naeja-RGM Pharmaceuticals (Grant/Research Support)Nosopharm SAS (Grant/Research Support)Novartis Pharmaceuticals Corp. (Grant/Research Support)NuCana Biomed (Grant/Research Support)Paratek Pharmaceuticals, Inc. (Grant/Research Support)Polyphor, Ltd. (Grant/Research Support)Prothena Corporation (Grant/Research Support)PTC Therapeutics (Grant/Research Support)Rempex Pharmaceuticals (Grant/Research Support)Roche TCRC (Grant/Research Support)Sagimet (Grant/Research Support)scPharmaceuticals Inc. (Grant/Research Support)Scynexis (Grant/Research Support)Spero Therapeutics (Grant/Research Support)TauRx Therapeutics (Grant/Research Support)Tetraphase Pharmaceuticals (Grant/Research Support)Theravance Biopharma Pharmaceutica (Grant/Research Support)USCAST (Grant/Research Support)VenatoRx (Grant/Research Support)Vical Incorporated (Grant/Research Support)Wockhardt Bio AG (Grant/Research Support)Zavante Therapeutics (Grant/Research Support)Zogenix International (Grant/Research Support) Vipul Kumar, PhD, Spero Therapeutics  Sujata M. Bhavnani, Pharm.D., M.S., FIDSA, 3-V Biosciences (Grant/Research Support)Achogen (Grant/Research Support)Amplyx Pharmaceuticals, Inc. (Grant/Research Support)Arixa Pharmaceuticals (Grant/Research Support)Arsanis Inc. (Grant/Research Support)B. Braun Medical Inc. (Grant/Research Support)Basilea Pharmaceutica (Grant/Research Support)BLC USA (Grant/Research Support)Boston Pharmaceuticals (Grant/Research Support)Bravos Biosciences, LLC Cidara Therapeutics Inc. (Grant/Research Support)Cipla, USA (Grant/Research Support)Corcept Therapeutics (Grant/Research Support)Cumberland Pharmaceuticals (Grant/Research Support)Debiopharm International SA (Grant/Research Support)Discuva Limited (Grant/Research Support)Emerald Lake Technologies (Grant/Research Support)Enhanced Pharmacodynamics (Grant/Research Support)Entasis Therapeutics (Grant/Research Support)E-Scape Bio (Grant/Research Support)Genentech (Grant/Research Support)Geom Therapeutics, Inc. (Grant/Research Support)GlaxoSmithKline (Grant/Research Support)Hoffmann-La Roche (Grant/Research Support)Horizon Orphan LLC (Grant/Research Support)ICPD Biosciences, LLC Indalo Therapeutics (Grant/Research Support)Insmed Inc. (Grant/Research Support)Institute for Clinical Pharmacodynamics (Employee)Iterum (Grant/Research Support)KBP Biosciences USA (Grant/Research Support)Kyoto Biopharma, Inc. (Grant/Research Support)Matinas (Grant/Research Support)Meiji Seika Pharma Co., Ltd. (Grant/Research Support)Melinta Therapeutics (Grant/Research Support)Menarini Ricerche S.p.A. (Grant/Research Support)Merck & Co., Inc (Grant/Research Support)Mutabilis (Grant/Research Support)Nabriva Therapeutics AG (Grant/Research Support)Naeja-RGM Pharmaceuticals (Grant/Research Support)Nosopharm SAS (Grant/Research Support)Novartis Pharmaceuticals Corp. (Grant/Research Support)NuCana Biomed (Grant/Research Support)Paratek Pharmaceuticals, Inc. (Grant/Research Support)Polyphor, Ltd. (Grant/Research Support)Prothena Corporation (Grant/Research Support)PTC Therapeutics (Grant/Research Support)Rempex Pharmaceuticals (Grant/Research Support)Roche TCRC (Grant/Research Support)Sagimet (Grant/Research Support)scPharmaceuticals Inc. 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Neuroendocrine neoplasms (NENs) are a complex group of tumours that occur in many organs. Routinely used IHC markers for NEN diagnosis include CgA, synaptophysin, Ki67 and CD56. These have limitations including lack of correlation to clinical outcomes and their presence in non-tumour tissue. Identification of additional markers and more quantitative analyses of tumour tissue has the potential to contribute to improved clinical outcomes. We used qRT-PCR to profile the expression levels of a panel of markers in tumour and matched non-tumour tissue from a patient with a G1 pancreatic neuroendocrine tumour. Differences in mRNA levels between tumour and non-tumour tissue were compared with IHC analyses of the same sample.An elderly man presented with lower abdominal pain for 6 months. Histological analysis identified a low grade, well differentiated pancreatic endocrine neoplasm. Twenty-seven tumour markers for neuroendocrine status, proliferation, stem cell phenotype, angiogenesis, epithelial to mesenchymal transition, cell adhesion, differentiation and tumour suppression were selected from previous studies and mRNA levels of these markers were measured in tumour and adjacent non-tumour tissue sample using qRT-PCR. IHC was carried out on the same tissue to detect the corresponding marker proteins. Of the markers analysed, seven showed higher mRNA levels in tumour relative to non-tumour tissue while thirteen had lower expression in tumour relative to non-tumour tissue. Substantial differences in mRNA levels were a gain of CgA, CD56, β-catenin, CK20, PDX1 and p53 and loss of Ki67, PCAD, CK7, CD31, MENA, ECAD, EPCAM, CDX2 and CK6. Comparison of qRT-PCR data with IHC showed correlation between fifteen markers.Our study is unique as it included matched controls that provided a comparative assessment for tumour tissue analysis, whereas many previous studies report tumour data only. Additionally, we utilised qRT-PCR, a relatively quantitative diagnostic tool for differential marker profiling, having the advantage of being reproducible, fast, cheap and accurate. qRT-PCR has the potential to improve the defining of tumour phenotypes and, in combination with IHC may have clinical utility towards improving tumour stratification or distinguishing tumour grades. The results need to be validated with different grades of NENs and related to clinical outcomes.The online version contains supplementary material available at 10.1186/s12876-021-01705-9. Neuroendocrine neoplasms (NENs) are a heterogeneous group of malignancies that can occur in all organs of the body, with the gastrointestinal system being a common site of localisation. Within the GI tract, the most common sites are the small intestine, rectum, colon, stomach, pancreas and appendix . NENs exGastrointestinal NENs are clinically challenging in their management. Some patients present with circulating levels of biologically active peptides including circulating chromogranin A and serotonin while others, termed non-functional NENs may have no specific clinical indicators but present with local symptoms including obstruction and bleeding . Non-funHistological morphology and immunohistochemical criteria are routinely used to diagnose NENs. Established diagnostic immunohistochemical markers include Ki-67 a marker of proliferation, chromogranin A (CgA), synaptophysin, and CD56 , 9, cytoBased on the 2019 WHO classification , NENs arIn a study of 200 patients with pancreatic endocrine carcinoma, the Ki-67 index was the major risk factor in relation to disease progression . While tA lack of reliable predictive and prognostic markers in neuroendocrine neoplasms on which to base therapeutic choices was identified by the ENETs , 17. SinThe aims of this study were to use RT-qPCR to measure mRNA levels of twenty-seven markers in a pancreatic G1 NET tumour relative to matched non-tumour tissue, to obtain a marker profile that could have clinical utility towards improving stratification of G1 pancreatic tumours or distinguishing tumour grades.A 66 year-old gentleman presented to his chiropractor with lower back pain having been asymptomatic for the previous 6 months. An ultrasound scan of his kidneys and a subsequent CT KUB + Abdo/Pelvis showed two solid lesions at the mid pole and lower pole of the right kidney and one heterogeneous solid lesion at the lower pole of the left kidney. It also showed a lesion in the pancreatic head measuring 3 cm with a dilated pancreatic duct and two small lymph nodes in the periportal region.A CT scan of the chest was done to complete staging and this showed a 3 mm nodule in the inferior segment of the lingula. This was indeterminate and too small to be characterized. Apart from that, his CT of the abdomen and pelvis was reviewed and this confirmed two solid lesions in the right kidney and also one in the left kidney. The pancreatic head lesion was also identified with the two small lymph nodes in the peri-portal region. In terms of blood tests, his tumour markers were all within the normal limits. This included a CA19.9 of 32, CEA 1.8, PSA < 0.1 and alpha feta protein of 4.An open distal pancreatectomy was carried out. After two years there was no clinical, radiological or biochemical evidence of recurrence of the neuroendocrine tumour.Histopathological analysis identified a well differentiated pancreatic endocrine neoplasm, low grade which was encapsulated on the external surfaces. However, elsewhere within the pancreas, tumour had infiltrated into the adjacent exocrine gland. The tumour had an organoid growth pattern with tubular, alveolar and trabecular groupings separated by a fibrous stroma. Cells showed mild nuclear pleomorphism with small nucleoli and gritty chromatin with abundant granular cytoplasm. Mitoses were not seen in 50 HPF and no necrosis was present. Ki-67 staining was variable with generally less than 2% staining positive. No perineural or lymphovascular space invasion was identified. The resections margins were free of tumour with 15 mm of clearance at the proximal pancreatic margin and not present on the inked external surface which was separated from the tumour by 1 mm thick fibrous capsule. A diagnosis of well differentiated pancreatic endocrine neoplasm, low grade pT2NX, was made.Regions of patient tissue showing normal pancreatic morphology were classed as non-tumour and regions where tissue was disorganised, containing cells with irregular nuclei and heterogeneity of size were classified as tumour tissue .Twenty-seven markers previously associated with a range of carcinoma characteristics with purported preventive or prognostic value were selected for the current study. Table mRNA transcript levels of each marker were measured in tumour relative to matched non-tumour tissue  and Olympus DP software was carried out for each of the twenty-seven markers in tumour and non-tumour tissue  to measure the mRNA levels of twenty-seven cancer markers associated with proliferation, metabolic activity, invasive potential and metastasis, in tissue from a patient with a G1 pancreatic NET. Comparison of mRNA marker levels from tumour tissue with adjacent non-tumour tissue from the same patient provided novel insights into the changes associated with acquisition of the cancer phenotype. The inclusion of matched controls enabled a comparative analysis, in comparison to many previous studies reporting tumour data only, which has limited value by itself when the status of the non-tumour tissue is unknown. Of the twenty-seven markers, CgA had a transcript abundance that was 80 times higher in tumour relative to non-tumour. CD56 and p53 were 6.5-fold more abundant in tumour relative to non-tumour tissue and β -catenin, PDX1 and CK20 were 2–3 times as abundant in tumour than non-tumour tissue. The profile of the tumour tissue indicated an overall reduction in mRNA levels of the analysed markers relative to non-tumour tissue. Ki67, PCAD and CK7 expression was reduced up to tenfold in the tumour relative to non-tumour with 2—ninefold reductions in CD24, CD44, CD31, CD49 MENA, ECAD, EPCAM, CDX2, CK6 and CK13. This suggests an overall loss of activity of these genes in the tumour. The most distinctive features of the tumour tissue were a gain in CgA, CD56, p53, PDX1, CK20 and a loss of Ki67, PCAD and CK7.The observed increases in mRNA transcripts of the mesenchymal markers vimentin and β -catenin, decreased transcripts of the cell adhesion markers E-cadherin and P-cadherin and loss of differentiation markers CDX2, CK6, CK7 and CK13 are hallmarks of transition to a mesenchymal phenotype tumour cells. The epithelial to mesenchymal transition is a process by which normal epithelial cells lose their cell:cell and cell:matirix adhesion and acquire the migratory and invasive characteristics of a cancer cell .This study indicates that qRT-PCR can distinguish phenotypic differences between tumour and non-tumour tissue. Advantages of qRT-PCR are that it is a semi-quantitative tool that generates numerical values for marker expression so comparisons can be made between different patients, as each marker is measured relative to a housekeeping gene. Other benefits are that it is quick and multiple markers can be tested in a single run. qRT-PCR is cheap, requiring only a few enzymes, buffers and primers. The same set of identical primers can be used in multiple labs thereby reducing variabilities between labs. A further advantage of qRT-PCR is that the RNA is extracted from the whole tissue and same extract used for all marker analyses. The extract is representative of the whole tissue rather than a portion of the tissue. PCR avoids the use of an antibody and the variability associated with antibodies to the same protein but from different sources.qRT-PCR measures mRNA levels markers relative to a housekeeping gene or control gene. This is selected to be stably expressed under different experimental conditions and for its expression to remain constant in different tissues and not influenced by the presence of disease so the expression of other genes can be measured relative to it. β-actin and GAPDH are commonly used housekeeping genes as their expression has been found to remain constant under different conditions , 20. Howand selected the one with least variation. We found β-actin to have less variation between tumour and non-tumour, compared to GAPDH for the pancreatic NET sample. Therefore, β-actin was selected as the housekeeping gene for analysis and future experiments.To provide a profile of the different markers expressed in tumour and non-tumour tissue, we measured mRNA levels of markers in each of these tissues relative to each other using DAXX as a reference as this gene was expressed in similar levels in tumour and non-tumour. This data showing the pattern of expression of different markers in tumour and non-tumour tissue provides a profile that may have application in defining individual patients. CgA for example is seen to be significantly low in the normal tissue profile while it has a massive increase in the tumour tissue. Future studies are required to validate these data using a larger number of patients and determine whether such profiles have potential for stratification of NEN tumours. A larger cohort may establish the possibility to Identify markers that are evidently different between normal and tumour to draw out a reference profile for different grades of NETs.We compared qRT-PCR results with IHC. Fifteen of the twenty-seven markers showed the same expression trends between tumour and non-tumour tissue and twelve showed differences in trends between tumour and non-tumour tissue. The most notable differences between the qRT-PCR and the IHC results were Ki67, MENA, ECAD and the cluster of differentiation markers CD24, CD44 and CD31. Our data showed Ki67 was higher in tumour relative to non-tumour using IHC but qRT-PCR analysis indicated Ki67 mRNA was reduced in tumour tissue relative to the non-tumour. Previous studies comparing Ki67 protein with Ki67 mRNA levels indicated variable correlations amongst different tumour samples . VariabiOur study showed that for four of the seven CD markers , the mRNA and protein data did not correlate. This is consistent with a previous study where concordance between gene and protein expression for a range of CD antigens in normal and prostrate tumour samples was poor to moderate (Pearson correlations ranged from 0 to 0.63), attributed to low levels of protein expression, sample preparation as well as the real biological differences between protein and mRNA expression . CD24 fuSynaptophysin was not detected by IHC and was only barely detectable by qRT-PCR, showing a slight increase in tumour relative to non-tumour. The levels of this transcript may not have been sufficient to produce detectable levels of protein. IHC staining for synaptophysin varies between different NENs and was reported as positive for approximately 60% of gastrointestinal neuroendocrine carcinomas . EPCAM mIHC is the traditional method for tumour diagnosis, essential for establishing tissue morphology and providing information for grading of tumour biopsies. IHC provides information about morphology of tissue that cannot be obtained with PCR. IHC is a qualitative and relatively time-consuming procedure in which the scoring technique may dependent on the scorer. Small changes of a few percentage in the Ki67 index can affect the grading of the tumour which itself may have an influence on the type of treatment regime chosen. While the new WHO NEN guidelines have improved the stringency of IHC methods , the hetOur study provides a unique analysis for NEN tissue relative to other reported studies, through utilization of the technique of PCR and inclusion of control tissue for comparison to the tumour tissue. The panel of markers screened includes a wide range that encompasses many tumour characteristics. PCR analysis of a G1 pancreatic sample revealed differential expression of 20 markers between tumour and matched normal tissue, out of a total of 27 markers tested. That many markers were found to be differentially expressed between tumour and non-tumour indicates marker profiling as a potential, additional diagnostic tool for better defining and stratification of tumours. As this was a pilot study based on a single NET case, the utility of the approach to measure multiple markers requires validation in a cohort study of different NEN grades and correlation with clinical outcomes. A better understanding of tumour cell phenotypes and their cell biology will contribute to improved clinical outcomes for patients.Additional file 1: Detailed materials and methods.


Primary sclerosing cholangitis (PSC) is in 70% of cases associated with inflammatory bowel disease. The hypermorphic T108M variant of the orphan G protein-coupled receptor GPR35 increases risk for PSC and ulcerative colitis (UC), conditions strongly predisposing for inflammation-associated liver and colon cancer. Lack of GPR35 reduces tumour numbers in mouse models of spontaneous and colitis associated cancer. The tumour microenvironment substantially determines tumour growth, and tumour-associated macrophages are crucial for neovascularisation. We aim to understand the role of the GPR35 pathway in the tumour microenvironment of spontaneous and colitis-associated colon cancers.Mice lacking GPR35 on their macrophages underwent models of spontaneous colon cancer or colitis-associated cancer. The role of tumour-associated macrophages was then assessed in biochemical and functional assays.Gpr35 in macrophages profoundly reduces tumour growth in inflammation-associated and spontaneous tumour models caused by mutant tumour suppressor adenomatous polyposis coli. Neoangiogenesis and matrix metalloproteinase activity is promoted by GPR35 via Na/K-ATPase-dependent ion pumping and Src activation, and is selectively inhibited by a GPR35-specific pepducin. Supernatants from human inducible-pluripotent-stem-cell derived macrophages carrying the UC and PSC risk variant stimulate tube formation by enhancing the release of angiogenic factors.Here, we show that GPR35 on macrophages is a potent amplifier of tumour growth by stimulating neoangiogenesis and tumour tissue remodelling. Deletion of Activation of the GPR35 pathway promotes tumour growth via two separate routes, by directly augmenting proliferation in epithelial cells that express the receptor, and by coordinating macrophages’ ability to create a tumour-permissive environment. A coding variant (T108M) in the G protein-coupled receptor GPR35 increases the risk for primary sclerosing cholangitis (PSC) and ulcerative colitis (UC). PSC and UC are chronic inflammatory conditions that significantly increase the risk for the development of cancer, and GPR35 is highly expressed in colorectal cancer.T108M is hypermorphic and promotes the pump’s ion exchange and signal transduction function resulting in increased proliferation of intestinal epithelial cells. Mice lacking Gpr35 globally or conditionally in their Vln+ cells  develop reduced numbers of tumours in sporadic cancer and colitis-associated cancer.GPR35 interacts with the sodium-potassium pump (Na/K-ATPase). Na/K-ATPase is the quintessential controller of a cell’s electrochemical gradient and controls Src-kinase signalling. The UC/PSC-associated GPR35Gpr35 in LysM+ cells reduces tumour growth substantially. GPR35 on macrophages increases the secretion of the angiogenic factors CXCL-1 (the murine orthologue of interleukin 8 (IL-8)) and VEGF. GPR35 on LysM+ cells promotes vessel sprouting from ex vivo cultured aortic rings which is dependent on its interaction with the Na/K-ATPase. Loss of GPR35 on macrophages reduces matrix metalloproteinase activity in tumour tissue, a prerequisite for tissue remodelling and angiogenesis. Macrophages carrying the human risk variant GPR35T108M secrete substantially more VEGF, IL-8 and matrix-metallo-proteinases (MMP2). Risk variant supernatants induce tube formation in human microvascular endothelial cells. Pharmacological pepducin-based GPR35 blockade markedly reduces tumour size and tissue levels of CXCL-1, VEGF, MMP2 and MMP9. GPR35 in macrophages is an upstream central coordinator of tissue remodelling and tumour-promotion, creating a tumour-permissive environment.Tumours lacking GPR35 are smaller compared with wild-type tumours. Deletion of Anti-VEGF antibodies are approved in cancer therapy to reduce tumour growth and angiogenesis, anti-IL-8 antibodies are in phase 2 clinical trials and have huge promise to slow down tumour angiogenesis.Blocking GPR35 could be very promising in cancer therapy as this reduces the release of both, VEGF and IL-8 and reduces tumour cell proliferation by inhibiting neoangiogenesis.APC) gene are a key event in both spontaneous and hereditary CRC.Colorectal cancer (CRC) is the leading cause of death from gastrointestinal malignancy, and the second most common cause of cancer-related death in the western world.GPR35  is associated with risk for PSC and UC.2+ homoeostasis. The UC/PSC-associated GPR35T108M variant is hypermorphic in its stimulatory activity on the pump.Gpr35 develop fewer tumours in a sporadic murine cancer model  and in a model of CAC induced by the mutagen azoxymethane followed by dextran sodium sulphate (AOM-DSS).Gpr35 selectively in the intestinal epithelium was sufficient to reduce tumour numbers.et al report that LPA activation of GPR35 in CX3CR1+macrophages maintains tumour necrosis factor-mediated intestinal homoeostasis and thereby reduces acute inflammation.A coding variant in Mutations in oncogenes and tumour-suppressor genes lead to unorganised proliferation and reduced cell death which initiates the development of malignancies. Tumour growth is not only determined by signal transduction pathways leading to proliferation,Atypical angiogenesis does not only play an important role in tumour neo-vascularisation,min) and CAC (AOM/DSS) models. We demonstrate that deletion of Gpr35 in macrophages recapitulates the profoundly reduced tumour size observed in Gpr35–/– germline mutant mice, identifying macrophage GPR35 as a key driver of tumour growth. Aortic tissue from mice lacking GPR35 is strikingly less capable of developing vascular sprouts. Vessel sprouting is mainly dependent on GPR35’s interaction with the Na/K-ATPase-dependent activation of Src kinase, and does not require ligand-induced GPR35 activation.Here, we report that macrophage GPR35 is important in vessel growth in healthy and malignant tissue. GPR35 critically controls the size of intestinal tumours in murine spontaneous  and CAC (AOM/DSS) models.Gpr35 in the intestinal epithelium  was sufficient to reduce tumour incidence in both models.Gpr35–/– mice were strikingly smaller compared with Gpr35+/+ tumours  and compared them to their Gpr35-sufficient controls   to AOM/DSS colitis revealed a similarly stark reduction in tumour size compared with their Gpr35fl/fl controls  DSS, nor during the three cycles of AOM/DSS was indifferent between Gpr35+/+ and Gpr35–/–, and Gpr35fl/fl and Gpr35ΔMΦ mice  . Gpr35Δmin mice , althougmin mice . Subjectcontrols . In this/fl mice , and ass/fl mice . The redΔMΦ mice . Levels /fl mice . Inflamm tumours . MacrophΔMΦ mice . Of noterophages . Neutrop context . Altoget10.1136/gutjnl-2020-323363.supp2Supplementary dataGpr35fl/fl mice appeared to be different from that in tumours of Gpr35ΔMΦ mice. We observed fewer endothelial cells, identified by CD31, which stretched along the adenomatous crypts in Gpr35ΔMΦ mice, whereas in adenomas from Gpr35fl/fl mice, the CD31+ neo-vasculature resembled a reticular structure , or the murine endothelial cell lines SVEC40 and H2-11 expressed Gpr35 mRNA , addition of APMA to supernatants revealed substantial activity in Gpr35+/+ and much lower activity in Gpr35–/– macrophage supernatants  reduced the number of inflammatory colon tumours.f ERK1/2 . Furtherf ERK1/2 . AltogetGpr35 identifies a novel major mechanism, and vividly highlights the critical role of myeloid cells in tumour development.Gpr35 reduces intestinal tumour incidence and reduces epithelial cell proliferation.Here, we report that GPR35 on macrophages is a major promoter of tumour growth by facilitating tumour neovascularisation. We show that this activity is dependent on Na/K-ATPase, and Na/K-ATPase-dependent activation of the non-receptor tyrosine kinase Src within macrophages. The profound reduction in tumour growth observed in both spontaneous and inflammation-induced intestinal tumour models on macrophage-selective deletion of Gpr35 deletion are smaller, contain fewer CD31+ endothelial cells and CD206+ M2-like TAMs, and exhibit lower expression of VEGF and CXCL-1, altogether implying reduced angiogenesis as a major component of reduced tumour growth when GPR35 is absent. Enhanced monocyte endothelial transmigration in vitro, and aortic sprout formation ex vivo, further corroborate the critical role of macrophage GPR35 in coordinating an angiogenic response. MMPs, in particular MMP-2, MMP-9 and MMP-14,Gpr35–/– tumours corroborates a role of this GPCR as an upstream regulator of tumour tissue remodelling.Neoangiogenesis is imperative for tumour growth and progression. This is therapeutically exploited by VEGF neutralisation (eg, bevacizumab in CRC2+, which is maintained via secondary-active transport, to levels otherwise only observed on receptor-mediated signalling events.2+ in turn affects Ca2+-dependent signalling processes broadly.GPR35 forms a complex with Na/K-ATPase and promotes its pumping activity, independent of pharmacological or candidate physiological ligands.In summary, we identify GPR35 on myeloid cells as upstream coordinator of tumour tissue remodelling, reinforcing the critical role of TAMs and neutrophils in cancer progression. The GPR35–Na/K-ATPase–Src pathway is precisely targetable with GPR35-selective pepducins.