Inappropriate testing remains a high healthcare cost driver. Tumour marker tests are more expensive than routine chemistry testing. Implementing test demand management systems like electronic gatekeeping (EGK) has reportedly decreased test requests.This study aimed to describe the appropriateness of tumour marker tests, carcinoembryonic antigen, alpha foetal protein, prostate-specific antigen, carbohydrate antigen 19-9, cancer antigen 15-3, cancer antigen 125, and human chorionic gonadotropin, and determine the effectiveness of the EGK used in the public health sector in KwaZulu-Natal, South Africa.Tumour marker test data for the KwaZulu-Natal province were extracted from the National Health Laboratory Service Central Data Warehouse for 01 January 2017 – 30 June 2017 (pre-EGK) and 01 January 2018 – 30 June 2018 (post-EGK implementation). Questionnaires were sent to the clinicians in the regional hospitals ordering the most tumour marker tests to assess ordering practices. In addition, we assessed monthly rejection reports to determine the effect of the EGK.The EGK minimally reduced tumour marker requests or associated costs (1.4% average EGK rejection rate). An overall 18% increase in the tumour marker tests occurred in 2018. The data suggest inappropriate tumour marker test utilisation, particularly for screening.The introduction of EGK as a test demand management had little impact on tumour marker test requests and costs. Continuous education and reiteration of indications for tumour marker test use are required.This study demonstrates the ineffectiveness of EGK in tumour marker orders, and provides some insight as to why these markers are being ordered, which is important in trying to decrease inappropriate ordering of these tests. However, the diagnostic sensitivity and specificity of most currently available serum tumour markers are limited.2Serum tumour markers are biochemical markers released by tumour cells directly or indirectly as a source or effect of malignant development. Tumour markers are a less invasive tool than a biopsy and are used to increase or decrease the clinical suspicion of a developing new or secondary cancer, detect the recurrence or progression of cancer, monitor response to treatment, and identify a specific therapeutic modality. Ideally, requesting and testing a tumour marker should allow for effective patient management, when appropriately performed, and reduce unnecessary redundant costs.5 The improved analytical sensitivity and specificity of high-output automated platforms have increased accessibility to the use of tumour markers and increased the use of serum tumour markers.6 However, the progression in instrumentation has been incongruent with the adoption of evidence-based guidelines to guide the appropriate use of tumour markers.7The inappropriate use of serum tumour markers has been reported.8 Increased healthcare costs with decreased healthcare budgets have forced laboratories to develop strategies to reduce and prevent inappropriate testing.9The cost of inappropriate testing of tumour markers indirectly affects patient safety, depending on the management strategies initiated based on the results reported.9Demand management is one strategy that focuses on ensuring appropriate requests while ensuring quality care to the patient. Laboratories have adopted several strategies to limit test demand, such as requiring requesting physicians to have a predetermined educational level, redesigning test request forms, and physical and electronic-based gatekeeping and reflex testing.10 Some strategies have been validated for serum tumour marker testing,11 such as the minimum retesting interval (MRI). The MRI strategy stipulates the minimum time before repeating a test based on the test’s properties, such as clinical indication.13Most serum tumour markers are not recommended as first-line rule-in or rule-out tests for cancer, but rather for detecting tumour recurrence and monitoring treatment. Thus, demand management strategies have been developed.The South African National Department of Health, in conjunction with the National Health Laboratory Service, which serves the South Africa public health sector facilities, have used the MRI strategy, termed electronic gatekeeping (EGK), to manage test demand since 2017. Electronic gatekeeping was introduced to limit healthcare spending on ‘unnecessary’ laboratory investigations. The criteria for selecting the MRI were based on a combination of literature and consensus agreement between expert clinicians representing the Department of Health in each region, and expert pathologists.14Electronic gatekeeping implementation studies have demonstrated that EGK is an effective cost-saving tool for several laboratory tests. In 2010, Tygerberg Hospital management and the National Health Laboratory Service conducted a pilot project in Cape Town, South Africa, to identify the number of EGK-rejected and EGK-restored  tests, the costs saved, and the impact test rejections. The study concluded that the EGK was an effective and sustainable demand management tool. They found that most rejected tests were not restored, revealing the inappropriateness of those test requests. The use of EGK did not appear to negatively impact patient care but was an effective cost-saving tool.15However, an academic hospital in Gauteng province, South Africa, reported that EGK test demand management does not dramatically influence requesting behaviour or save costs. They reported an unchanged monthly percentage of EGK-held tests over a 22-month retrospective study period, suggesting that a solitary demand management strategy is not as effective as anticipated or as demonstrated in other studies.Both the Cape Town and Gauteng studies only reviewed the effect of routine chemistry testing demand, not tumour marker testing. To date, no study has reviewed the requesting nature of tumour markers in the South African public health sector. In the South African public healthcare sector, all laboratory and other diagnostic costs are borne by the state.Tumour markers were chosen as they are one of the most highly requested tests in the chemistry laboratory, are more costly to process, and are thus charged at a higher rate than the more routine general chemistry testing. At the time of the study, the National Health Laboratory Service Chemical Pathology laboratory at Laboratory A provided tumour marker testing services for most patients in the public sector covering the entire province of KwaZulu-Natal, except a more northern region, where testing is provided by Laboratory B, a National Health Laboratory Service laboratory, which provides a smaller tumour marker test repertoire. The serum tumour markers that were assessed during this audit were carcinoembryonic antigen, alpha foetal protein (AFP), prostate-specific antigen (PSA), carbohydrate antigen 19-9 (CA 19-9), cancer antigen 15-3 (CA 15-3), cancer antigen 125 (CA 125), and human chorionic gonadotropin (HCG).17 Carcinoembryonic antigen is a tumour marker for gastrointestinal cancers, but it is also elevated in breast, lung and liver cancers, and non-malignant conditions like heavy smoking, bronchitis, gastritis, duodenal ulcer, liver diseases, pancreatitis, and colorectal polyposis.18 Human chorionic gonadotropin is produced by embryonal tissue1 but is used as a tumour marker in seminomatous and non-seminomatous testicular tumours, ovarian germ cell tumours, the gestational hydatid form mole, choriocarcinoma, and non-testicular teratomas.19 Carbohydrate antigen 19-9 is normally synthesised by the pancreas, biliary ductal cells, gastric, colon, and endometrial and salivary epithelia. It is mainly used to prognosticate and monitor response to interventions in patients with pancreatic and gastrointestinal cancer.1 Increased CA 125 values most often are associated with epithelial ovarian cancer, although levels can also be increased in other malignancies, such as breast, endometrial, cervix, peritoneal, uterus, lung, pancreas, hepatocellular and non-Hodgkin’s lymphoma and multiple benign disorders, which include pregnancy, endometriosis, uterine fibroids, pancreatitis, normal menstruation, pelvic inflammatory disease, and cirrhosis of the liver.1 Cancer antigen 15-3 levels have been reported to be useful to prognosticate in breast cancer patients,20 but elevations of CA 15-3 levels are also seen in other malignancies, including pancreatic, lung, ovarian, colon, and liver cancer as well as benign breast and liver conditions.1 Prostate-specific antigen aids in the diagnosis, risk assessment, and monitoring of prostate carcinoma, but it is also elevated in non-malignant conditions like acute urinary retention, benign prostatic hyperplasia, prostatitis, and urinary tract infection.1Disorders with high AFP include hepatocellular carcinoma, hepatoblastoma, non-seminomatous testicular germ cell tumours of the embryonal carcinomas, cancers of the pancreas, lung, and gastric, and non-malignant processes such as acute viral hepatitis, liver cirrhosis, and obstructive jaundice.This study aimed to describe the tumour marker requesting practices across different levels of healthcare in the province of KwaZulu-Natal, South Africa, assess the effect of EGK on these requesting practices and, lastly, determine via questionnaire the rationale for tumour marker requesting by the clinicians at the highest ordering facilities.Ethics approval for this study was obtained from University of KwaZulu-Natal Biomedical Research Ethics Committee (number BE035/18). Written informed consent was received from the participating clinicians. Data were collected on a password-protected computer and the primary investigator was the only person with access to it. Patients were identified by hospital numbers and their identities were not revealed. Questionnaires were anonymised and identified by numbers allocated to the specific sites. Survey respondents were assured raw data would remain confidential and would not be shared.The National Health Laboratory Service Central Data Warehouse reposits all test results generated by National Health Laboratory Service laboratories. We extracted from the National Health Laboratory Service Central Data Warehouse all tumour marker tests performed in public healthcare facilities in KwaZulu-Natal province, South Africa. The data extracted included requests made 6 months pre-EGK (01 January 2017 to 30 June 2017) and 6 months post-EGK implementation (01 January 2018 to 30 June 2018). The data consisted of results from Laboratory A and B National Health Laboratory Service Chemical Pathology laboratory. Laboratory A offers the following tumour marker tests: carcinoembryonic antigen, AFP, PSA, CA 19-9, CA 15-3, CA 125, and HCG, whereas Laboratory B offers all the above tests except CA 19-9. Both laboratories analysed tumour markers on the Siemens Advia Centaur XP . The MRI rules that were in use for EGK per tumour marker test were as follows: HCG: 1 day; AFP: 1 month; PSA: previous result abnormal = 1 month and previous result normal = 1 year; carcinoembryonic antigen: 1 month; CA 125: 1 month; CA 19-9: 1 month; and CA 15-3: 1 month; where: 1 day is 12 h, 1 month is 21 days, and 1 year is 322 days since daily tests are not performed at the same time each day and a repeat visit within a set interval (eg. week or month) may happen before the exact interval has passed. The monthly EGK rejections reports were assessed from 01 January 2018 to 30 June 2018, to determine the number of requests rejected by gatekeeping in the included laboratories.21) were hand delivered and obtained from clinicians from these selected facilities. The questionnaire identified who was ordering tumour markers and why. Responses were collated in Microsoft Excel  for further evaluation.The top five highest requesting units from all healthcare facilities over the period of the study were identified and chosen as the sites for questionnaire distribution. Written informed consent and the questionnaire  and Microsoft Excel 2016 . Data were assessed for normality using the Shapiro-Wilk test. Normal data were presented as mean ± standard deviation. The monthly average rejection rate was calculated. Costing was done using on the National Health Laboratory Service State Price List 2017.A total of 38 615 tumour marker tests for the specified analytes were processed during the 6-month pre-EGK introduction period (01 January 2017 to 30 June 2017), while 45 567 tumour markers requests were processed in the post-EGK implementation period (01 January 2018 to 30 June 2018). In 2018, there was an 18% increase in tumour marker tests processed. The most ordered tumour marker was PSA , while the least ordered was CA 15-3 (< 3% of tested tumour markers in both 2017 and 2018) .The majority of samples for PSA, AFP, carcinoembryonic antigen, CA 19-9, CA 15-3 and CA 125 had normal results  in both years of the study period. In contrast, HCG had the most abnormal results  for both years in the study period, with 90.1% (2783/3088) abnormal results for 2017, and 92.1% (3771/4092) for 2018.n = 21 299, 65%). A minority of samples  had a history of cancer documented on the request forms. Nine percent (n = 2927) of requests indicated suspicion of malignancy or screening as request reason  ($218 409.51 United States dollars [USD]), while abnormal results cost R1 210 461.00 ($66 167.61 USD). In the same period in 2018, normal results cost R4 764 052.00 ($260 418.09 USD) and abnormal results cost R1 181 010.00 ($64 557.73) .Most test requests were received from the outpatient departments or non-oncology clinics of the respective healthcare facilities. However, test requests from the oncology wards and clinics were the lowest. The tertiary academic hospitals made the fewest requests, followed by primary healthcare facilities. Requests from district hospitals increased in 2018 by 58.7% to overtake regional hospitals as the main requestors .The EGK rejected an average of 95 tumour marker test requests per month from 01 January 2018 to 30 June 2018 with a total of 570 tests rejected over this period. Additionally, during the same 6-month period, no EGK-rejected tumour marker tests were restored. The total cost of tumour marker rejected test requests was R78 043.86 in 2018 .n = 24; 64%), followed by medical , with the remainder  being from general outpatient clinics or unspecified. Participants consisted predominantly of junior staff (interns) and non-specialist medical officers.We reviewed 22 responses from the 37 questionnaires distributed (59% response rate). Most respondents were from surgical departments (n = 21) of respondents indicated that their facility had no dedicated oncology unit or clinics. A further 91% (n = 20) of the participants were unaware of any local or international tumour marker test request guidelines for clinical practice. Participants indicated the following as consequent actions to an abnormal tumour marker result: imaging studies , 63% (n = 14) included biopsy, referral , requesting another tumour marker test , and 9% (n = 2) included repeating the tumour marker test. Most respondents requested tumour markers to query suspected tumours; over 20% (n = 4) indicated their use to detect tumour sources . All rejected tumour marker tests were estimated to cost R78 043.86 ($4266.12 USD) in 2018. In the public sector in South Africa, the cost of laboratory testing is paid by the Department of Health (state), with no cost to the patient. The EGK rejects test requests before payment and laboratory testing, hence no refund is made on rejected requests.23 Pema, Kiabilua and Pillay ,15 and Smit, Zemlin and Erasmus 14 reported significant cost reductions through EGK of requests. However, the test requests reviewed were smaller-volume tests. Our findings showed that the number of tumour marker tests rejected by the EGK rules was minimal. Fewer than 20 tests were rejected on average, per month, for each of the tumour markers apart from PSA. This low rejection rate suggests that appropriate test ordering, per the test’s correct clinical requirement and guidelines, would be the most effective way of controlling inappropriate tumour marker test requests. Appropriate test ordering practice requires education on and routine reiteration of appropriate request guidelines, and the development and implementation of national testing guidelines. The lack of continuous clinician education is a reported driver of inappropriate testing.25 Education and continuous reiteration of best practices are especially important in the non-academic centres, where there are more generalists than specialists managing patients. The high request numbers from district health facilities support the fact that education regarding tumour marker utilisation is most needed in non-academic centres; however, district hospitals represent most hospitals servicing the population (n = 37) versus regional hospitals (n = 13).26 As evidenced by the respondents on the questionnaire, a lot of clinicians were requesting tumour markers to screen for malignancy.Previous reports from 1997–2012 state that 20% – 50% of laboratory tests are inappropriate or are not evidence-based practices.Our findings may also be an indication that the EGK rules require further review and are not strict enough to achieve sufficient demand management. These rules could include limiting requests to only two tumour marker tests on one visit or admission to dissuade panel screening. However, stricter rules may not be possible for HCG, as it is also a test for normal pregnancy and pregnancy-related disorders (for example ectopic pregnancy) and serial measurements are critical. Human chorionic gonadotrophin was frequently requested for younger patients, probably because the laboratory information system could not distinguish between malignancy-related and pregnancy-related HCG testing. Additionally, germ cell tumours in which HCG concentrations may be increased are more frequently seen in younger adults and adolescents.29This is the first study, to the authors’ knowledge, that examines the use of tumour markers in sub-Saharan healthcare facilities. The findings of this study suggest that repeat testing represents a small fraction of the cost associated with tumour marker requests and that inappropriate requests  are likely resulting in test overuse and associated increased healthcare costs. The introduction of EGK has made little or no impact on the number and cost of tumour marker tests performed. While there are no national consensus guidelines for the utilisation of tumour markers in South Africa, international guidelines or best practice documents are available to guide clinicians to order tests appropriately.We recommend developing local and national tumour marker ordering guidelines for all levels of healthcare. Focused education at the undergraduate level and continuous professional development regarding appropriate utilisation of laboratory tests including tumour markers is also required. Greater involvement of pathologists in spreading appropriate utilisation awareness and coaching of junior doctors is also essential.The increasing demands on limited healthcare resources and funding necessitate careful management of testing to ensure optimal patient care while managing costs.One of the limitations of this study was that access to histology results was not available. Thus, tumour marker test results could not be confirmed by tumour biopsy results. Furthermore, the small number of questionnaires distributed may not be representative of the clinician cohort. Additionally, we did not sample clinicians from different healthcare facility levels. Furthermore, due to the geographical limitations, lack of internet availability, and other limited resources, we restricted questionnaire distribution to facilities within KwaZulu-Natal. In addition to that, HCG results were not separated into pregnant versus non-pregnant due to missing clinical records on many samples. Lastly, the effects of interventions to improve clinician knowledge of tumour marker requests were not assessed.It appears that many clinicians do not appropriately request and utilise tumour marker tests and there is no guideline for tumour test ordering and result interpretation. The EGK barely reduced tumour marker requests and costs as there was an increase in costs and testing numbers, despite EGK implementation. Education of doctors, stricter EGK rules, and additional demand management measures may be required to make a noticeable demand difference.


To investigate the effect of IV fluid resuscitation on endothelial glycocalyx (EG) shedding and activation of the vascular endothelium and inflammation.A planned biomarker sub-study of the REFRESH trial in which emergency department (ED) patients) with suspected sepsis and hypotension were randomised to a restricted fluid/early vasopressor regimen or IV fluid resuscitation with later vasopressors if required . Blood samples were collected at randomisation (T0) and at 3 h (T3), 6 h (T6)- and 24 h (T24) for measurement of a range of biomarkers if EG shedding, endothelial cell activation and inflammation.p = 0.03), SYN-4  and IL-6 . No difference over time was observed between groups for the other biomarkers.Blood samples were obtained in 95 of 99 enrolled patients . Differences in the change in biomarker over time between the groups were observed for Hyaluronan (2.2-fold from T3 to T24, A consistent signal across a range of biomarkers of EG shedding or of endothelial activation or inflammation was not demonstrated. This could be explained by pre-existing EG shedding or overlap between the fluid volumes administered in the two groups in this clinical trial.Trial registration Australia New Zealand Clinical Trials Registry ACTRN126160000006448 Registered 12 January 2016.The online version contains supplementary material available at 10.1186/s40635-023-00508-4. Sepsis is defined as life-threatening organ dysfunction due to a dysregulated response to infection, with septic shock being a subset of sepsis in which underlying circulatory and cellular/metabolic abnormalities are profound enough to substantially increase mortality . The SurIn a landmark clinical trial of fluid bolus therapy in children with hypoperfusion in resource-poor settings in Africa, the excess mortality in those children treated with fluids was found to be due to cardiovascular collapse . This fiThe Restricted Fluid Resuscitation in Sepsis-associated Hypotension (REFRESH) trial was a multicentre randomised trial designed to determine the feasibility of comparing a fluid-restricted/early vasopressor regimen compared to usual care to achieve perfusion targets during the early resuscitation of patients with suspected septic shock in the emergency department . The aimThe methods for the REFRESH trial have previously been described , 20. BriResearch blood sampling was performed at randomisation and at 3, 6 and at 24 h later . Following collection, samples were processed within 2 h with serum collected by centrifugation at 3000 RPM for 10 min, followed by storage in 0.5 ml aliquots at − 80 °C. Samples were subsequently transported for batch analysis at the laboratory of the Centre for Clinical Research in Emergency Medicine in Perth, Australia. We selected the following biomarkers based upon prior work by our group and others. EG shedding: Syndecan 1 (SYN-1), Syndecan 4 (SYN-4), Hyaluronan, Heparan Sulphate. Endothelial Cell activation: Intercellular Adhesion Molecule (ICAM), Vascular Cell Adhesion Molecule (VCAM), E-Selectin, Vascular Endothelial Growth Factor Receptor-1 (VEGFR-1). Cardiac stretch: Pro-Atrial Natriuretic Peptide (Pro-ANP); Renal Injury: Neutrophil Gelatinase-Associated Lipocalin (NGAL); Systemic inflammation: Interleukin (IL)-6, IL-10, Resistin. Plasma biomarker concentrations were determined by enzyme-linked immunosorbent assay (ELISA) and Cytometric Bead Array (CBA) techniques. Heparan Sulfate was measured in plasma using a fully validated, commercial kit, with a lower limit of detection of 0.19 ng/ml . ICAM, VCAM, E-Selectin, IL-10 and IL-6 were measured in a multiplex CBA format using Flex sets . All remaining biomarkers were measured using DuoSet ELISA kits , and individually optimised prior to use to achieve average intra-plate coefficients of variation below 10%.p values < 0.1 were considered to provide sufficient evidence to warrant further consideration. In this instance, Bootstrapping was employed to investigate sensitivity to sampling variation.Biomarkers were analysed by group allocation according to intention to treat. Normality of distribution was assessed by inspection of box and whisker plots of values within assay limits by group at each timepoint. All variables demonstrated departures from normality and were subsequently log-transformed for analysis. An interaction of group and time was used in linear mixed models to assess whether the change over time in each marker differed between the groups. For biomarkers, where some values lay outside the limit of detection for the assay, random effects Tobit regression was used. These analysis techniques retain patients without biomarker data at all timepoints in the analysis by utilising maximum likelihood estimation (MLE). MLE uses all available information to calculate the most likely parameter estimates at the sample level as opposed to imputing values at the individual level. Results are reported as fold changes after exponentiating coefficients derived on the log scale. A per-protocol analysis was also undertaken excluding those cases in which there was a deviation from protocol regarding the recommended fluid volumes in each arm. As an exploratory study, no adjustment was made for multiple testing and interactions with p = 0.7. Table The REFRESH trial enrolled a total of 99 participants randomised to one of the two treatment arms . Mortalip = 0.04), driven by opposing slope directions between the two groups for T3 to T24 and T6 to T24. This resulted in a 2.2-fold change in slope for T3 to T24 (p = 0.03) and a 2.6-fold change for T6 to T24 (p = 0.01) for the restricted fluid group compared to usual care.A significant difference in Hyaluronan levels over time was detected  driven by a greater negative slope in the restricted fluid group compared to usual care, equating to a 1.6-fold change in slope between T3 and T24, p = 0.007 .There were no differences in absolute values at the T0 and T24 timepoints .There was no difference between the groups in the change over time for the natriuretic peptide Pro-ANP (interaction term p = 0.31), VCAM (p = 0.26), E-Selectin (p = 0.73), and VEGFR-1 (0.96).No differences between the groups were identified in the pattern over time of any biomarker associated with activation of endothelial cells. These were ICAM (p = 0.07) driven by a greater negative slope in the restricted fluids group  between T0 and T3 (p = 0.03) , Resistin (p = 0.43) and the renal injury biomarker NGAL (p = 0.15) over time.For the cytokine IL-6, the pattern over time differed between the groups . Bootstrapping of the analyses, where the interaction term In this planned sub-study of the REFRESH trial, differences over time in the pattern of Hyaluronan, SYN-4 and IL-6 were observed during the first 24 h of care driven by greater or contrasting downward patterns in the restricted volume group involving the T3 and T6 timepoints. No differences were noted for other biomarkers of EG shedding, SYN1, Heparan Sulphate, nor for Pro-ANP, IL-10 and any biomarkers related to endothelial cell activation.The question of the optimal approach to haemodynamic resuscitation has been identified as one of the top priorities for research in septic shock . The conPrevious studies have found evidence supporting an effect of IV fluids on the EG in sepsis. Pouska et al. demonstrated a persistent change in the perfusion boundary ratio on sublingual intravital microscopy, a surrogate measure of EG thickness, among 16 patients with sepsis undergoing fluid bolus administration . Smart ep = 0.07), in favour of the restricted fluid regimen [In a multicentre study of 619 patients with sepsis, there was no association between fluid volume and SYN-1 on the first day of admission, although SYN-1 was associated with illness severity and mortality . In a pr regimen . In our  regimen . The lacOverall, we found limited evidence for an effect of fluids on EG shedding. In support of the hypothesis are temporal changes in some biomarkers coinciding with the maximal difference in fluid volume during the first 6 h of treatment. Against is the lack of consistency across all EG and inflammatory markers, and the lack of any effect on changes in the endothelial activation biomarkers. Though there was a 20 ml/kg separation in fluid volume between the two study arms at 24 h, this difference may have been insufficient to yield a consistent effect on EG shedding. This is supported by there being no difference in Pro-ANP levels between the groups. In a preclinical endotoxic shock model, ANP was significantly higher in the fluid group ; howeverOf some interest is the pattern of IL-6 with a steeper decline in the restricted fluids group which coincided with the period of maximal difference in fluid volume. As an acute phase pro-inflammatory cytokine IL-6 displayed a typical pattern of decline over the first 24 h . The temIn critical illness, where EG shedding and inflammation are already established, identifying a consistent response to an intervention is challenged by the confounding effects of illness severity, the clinical heterogeneity of sepsis and the timing of presentation to hospital. In this pilot clinical trial set in the ED the overall severity of illness was lower compared to other sepsis studies. As a pragmatic clinical trial there were several protocol deviations which potentially impacted on separation; however, a ‘per-protocol’ analysis did not yield different results from the intention to treat analysis. Importantly, participants received at least 1 L of fluid prior to randomisation (Table One issue is the question of whether haemodilution may mask as significant difference in biomarkers measured between groups receiving different volumes of fluid. Some authors have adjusted biomarker results for albumin  or haemoThe question of whether exogenous intravenous fluid has a detrimental effect on EG shedding and inflammation in the setting of sepsis remains unclear. This study did not demonstrate a consistent signal across a range of biomarkers of EG shedding or of endothelial activation or inflammation. Patient heterogeneity, low illness severity and insufficient separation between the groups in the overall fluid volume mean that an effect cannot be excluded. While the results of clinical trials are awaited, a judicious approach to fluid management in sepsis is still recommended .Additional file 1. Analysis of biomarkers in those treated per protocol.Additional file 2. Analysis of biomarkers from refresh RCT.


Abdominal organs  are frequent targets of cancer cell invasion but their primary tumours are less known for their metastatic potential to other organs e.g. to the breast. Despite the known connection of the pathogenesis from breast cancer to liver metastasis, the study of the spread in the opposite direction has been neglected. The notion that breast cancer could be a metastasis besides being a primary tumour is based on rodents’ tumour models upon implantation of tumour cells under the capsule of the kidney or under the Glisson’s capsule of the liver of rats and mice. Tumour cells develop into a primary tumour at the site of subcutaneous implantation. The metastatic process starts with peripheral disruptions of blood vessels near the surface of primary tumours. Tumour cells released into the abdomen cross the apertures of the diaphragm, enter the thoracal lymph nodes and accumulate in parathymic lymph nodes. Abdominal colloidal carbon particles injected into the abdomen faithfully mimicked the migration of tumour cells and deposited in parathymic lymph nodes (PTNs). An explanation is provided why the connection between abdominal tumours and mammary tumours escaped attention, notably, parathymic lymph nodes in humans were referred to as internal mammary or parasternal lymph nodes. The apoptotic effect of Janus-faced cytotoxins is suggested to provide a new approach against the spread of abdominal primary tumours, and metastatic development. Molecules acting as double-edged swords, also referred to as Janus-faced molecules possess both beneficial and harmful biological effects. Due to their side effects, these compounds were not preferred in medical practice, which does not mean that their health benefits could not be utilized. A known double-edged sword is resveratrol, found in many plant species that belong to the polyphenol stilbenoids group containing two phenyl rings attached through an ethylene bridge . The polA classical example of a double-edged sword is cholesterol. High levels of cholesterol cause diabetes, high blood pressure, vascular and other diseases. Due to the low solubility, its high level may cause coronary heart attack that can lead to death. The amphipathic property and the shape of the cholesterol molecule make it suitable as a membrane insert . CholestAnother double-edged sword is nitric oxide (NO), one of the major air pollutants but also a gaseous signalling molecule playing an important role as a key vertebrate biological messenger –13. NitrThree types of antifungal agents have been reviewed recently, including double-edged mycotoxins, aminoglycoside molecules as double-edged swords, among them the most efficient minor fraction gentamicin B1, and the classical antifungal agents. Particular attention was given to the antifungal activities of these compounds in comparison with other classical antifungal drugs . The poisonous mycotoxins ubiquitous in nature are produced by various fungi species whose occurrence in the food chain is inevitable. Mycotoxins pose a serious problem on a global scale , 19 sincThe anticancer properties of double-edged compounds could be utilized for the benefit of patients when their survival is more important than the mutagenic side effects caused by these compounds. To decide which fungal toxins could be utilized alone or in combination with other antimetastatic agents, the side effects have to be clarified. The International Agency for Research on Cancer  distinguished three categories. Group 1) IARC compounds are carcinogenic to humans, and Group 2 compounds are probable carcinogens. Group 2A is probably carcinogenic to humans, group 2B is reasonably anticipated to be a human carcinogen. Group 3 is lacking carcinogenic properties, possesses tolerable side effects, and more importantly is among the candidates for antimetastatic agents. From the structure–function relationship of mycotoxins, only general conclusions have been drawn. Ring structures that contain heteroatoms, functional groups and the cumulative presence of oxygen atoms contribute to the oxidative stress and cytotoxicity of mycotoxins. The preselection of mycotoxins for medical treatment excludes category 1 and 2 IARC carcinogens, and only the IARC category 3 compounds have been considered as potential antitumour agents against the metastatic spread of abdominal tumours. By analogy in humans, a similar tumour spread could take place that could be prevented by apoptotic antineoplastic double-edged sword molecules. In conformity with this strategy in mind, the focus is placed on those mycotoxins and their precursors that besides being highly cytotoxic, attest to antitumour properties. No doubt that some of them will not only be considered antimetastatic agents but used to prevent patients diagnosed of having thoracal and mammary lymph node tumours. The cytotoxic side effect could induce mutagenesis that could develop into scondary tumours but later when the Janus-faced molecules already exerted their beneficial antineoplastic effects.Different types of antifungal agents have been reviewed recently, including the’double-edged’ mycotoxins, and aminoglycoside molecules as double-edged swords, among them gentamicin B1, the most efficient minor fraction of the gentamicin family. Particular attention was given to the antifungal activities, compared to classical antifungal drugs such as amphotericin B, clotrimazole, nystatin, and griseofulvin . Fungi cThe beneficial apoptotic effect of cytotoxicity is one of the potentials that can be utilized against the spread of cancer. Antifungal cytotoxins have been reluctantly applied against fungal infections as they raise ethical issues due to their mutagenic impact. Nevertheless, their utilization deserves consideration and could be useful not only for patients who suffer from fungal infections. This study suggests the extension of the application of Janus–faced molecules against breast cancer and mammary metastasis. Breast cancer patients are normally older than their fertility age. The short-term cytotoxicity of cancer treatment is likely to be favoured over the long-term mutagenicity. The short-term and beneficial anticancer treatment could induce secondary tumour development but years later of treatment when primary cancer or metastasis has been eradicated. Primary tumours were used to establish tumour cell lines that allowed to follow the development of metastasis in different organs and lymph nodes in the body of rodents (rats and mice). The establishment of tumour cell lines is unavoidable to secure the implantation of an exact number of cells and follow reproducibly the temporal aspects of the metastatic development. The preparation of tumour cell lines is schematically shown in Fig. We did not carry out research that involved human subjects. Regarding the ethical issue of animal experiments: rats and mice were kept in a conventional laboratory environment and fed on a semi-synthetic diet  and tap water ad libitum. The conditions under which animal experiments were performed are consistent with prevailing standards in the UK. Animals received human care to the criteria outlined in the Guide for the Care and Use of Laboratory Animals authorised by the Ethical Committee for Animal Research, University of Debrecen, Hungary (permission # 22). All experiments conformed to the European Convention for the protection of Vertebrate Animals used for Experimental and Other Scientific Purposes, 2010.Fusarium, Trichoderma, Trichthecium, Myrothecium, Trichothecium, Cephalosporium, Ventrici Monosporium. The most important structural elements of trichothecenes are the 12,13-epoxy rings responsible for the major toxicity, the presence of hydroxyl and acetyl groups and the side chains [- mycophenolic acid, penicillic acid, 5-methoxysterigmatocystin- anguidine analog scirpenol, di- and triacetoxyscirpenols- T-2 toxin and related trichothecenes- cytochalasin B- patulin- hydromytoxin B- 16-hydroxyroridin E- tenuazoic acid- betaacetoxyscirpendiol- gliotoxin- fluorinated pseurotin A- synerazol- rubratoxin B- beauvericin- the macrocyclic trichothecenes verrucarin A and roridin A- leuteoskyrin, a hydroxyanthraquinonederivativee related to MT81.The majority of the anticancer Janus-faced mycotoxins belong to the trichothecene analogues . Mycotoxe chains . The trie chains . Trichote chains –29. Mycoe chains , 31. Thee chains :- mycophThe efficient apoptotic effect of cytotoxicity seems to be suitable to be utilized against the spread of cancer. Despite some secondary metabolites reaching clinical trial phase II and more mycotoxin derivatives being expected to enter human clinical trials shortly, no fungi-derived agent has been approved as a cancer drug so far , 32. DesDue to the complexity of animal tumours, it is impossible to use a single solution and requires selection and application among models. The chicken chorioallantois-membrane model is among the oldest ones to study metastasis  but its The administration of tumour cells is prohibited in man, thus we have developed rat and murine models from N-dimethylnitrosamine–induced tumours, namely from mesenchymal nephroblastoma and hepatocarcinoma . The majSecondary tumour growth (metastasis) was measurable after 8 days of tumour cell implantation under the subrenal capsule or in the Glisson’s pocket of the liver of rats resulting in secondary tumours in the thoracal lymph nodes primarily in parathymic lymph nodes (PTNs). The initialhaematogenouss growth of the primary tumour is characterized by its hypoxic conditions and apoptosis inside the tumour, growth outward and disruption of the peripheral blood vessels due to the lagging angiogenesis and release of blood and tumour cells into the peritoneal cavity. The lymphatic part of the tumour spread starts when the increased volume of ascitic fluid and tumour cell accumulation in the peritoneal transudate forces tumour cells to cross the transdiaphragmatic pores and drain tumour cells into the thoracal lymphatic vessels and lymph nodes. Metastatic cells accumulate in the sentinel lymph nodes primarily in the internal mammary and parathymic lymph nodes . The accumulation of tumour cells could increase the volume of PTNs up to 70-times and their weight from 10 to 700 mg. India ink was implanted under the kidney capsule of rats and mice to mimic the lymphatic spread of colloidal particles in the thoracal lymph duct and nodes. The metastatic process was mimicked by the implantation and appearance of India ink colloidal particles in PTNs and confirmed the general mechanism from abdominal primary tumour growth through blood circulation and lymphatic development of metastasis in the thorax.18FDG-miniPET tumour diagnostic test has been adapted to investigate tumour growth in vivo in local and metastatic rat models. We have confirmed that (i) local s.c. administration of tumour cells generated local tumours unsuitable to follow the spread of metastasis (ii) Intravenous administration of tumour cell cause the unpredictable location of tumour formation, thus not regarded as a reliable metastatic tumour model. (iii) Subrenal and liver implantation proved to be suitable to follow the metastatic process in rats [The  in rats , 39. ThePrimary tumours metastasized from organs to other, often distant organs. The exception is the liver, which in this regard is not exactly known where it is projecting tumour cells. To estimate the metastatic potential of liver tumour slices or cells were implanted in the liver’s Glisson pocket and tumour growth is shown in Fig. The appearance of tumour cells in PTNs was also confirmed by the reimplantation of tumour-bearing parathymic lymph node slices. Reimplantation induced tumour growth under the subrenal capsule. Results corresponded to expectations, after one week of tumour growth there were sufficient number of metastatic cells in PTNs to reinduce primary tumour growth .The organs before the implantation of tumour cells are seen in Fig. PTNs in rats are located outside the thymus. In mic,e they are harboured inside the thymic capsule . Human pAfter the implantation of hepatocarcinoma (HeDe) rat tumour cells under the capsule, the bleeding of the retroperitoneal primary HeDe tumour indicated lagging angiogenesis and ruptures of blood vessels near the surface of the tumour  Schematic view of the thymus. RT, right lobe of the thymus; LT, left thymic lobe; IMN, internal mammary lymph node identical with PTN, parahymic lymph node; TC, thymic capsule. With permission [Saline (2 ml) without ink was administered ion Fig.  e.Controion Fig. f. PTNswermission .  and mesoblastic nephroma (NeDe) solid tumours  as well Despite their mutagenic impact,’double-edged’ molecules could help those patients who are older than their fertility age, and the anticancer treatment could be more important than the long-term mutagenic affect the development of which could take years or decades. Two groups of patients could be particularly interested in the treatment with Janus-faced anticancer agents. The incidence of breast cancer, the most common malignancy in women increases with age, with the patients diagnosed after menopause. In about 15–25% of cases, patients are premenopausal at the time of diagnosis of cancer, and about 7% are below the age of 40 .The treatment of postmenopausal breast cancer patients with Janus-faced anticancer cytotoxins is preferentially recommended, unlike prostate patients. Although the decline in sperm count, and quality  is measurable but men typically never stop producing sperm, thus the fertility age is less important than in women.Immunocompromised mouse strains allowed researchers to implant human tumour cells into mice without the risk of cell-mediated immunological tissue rejection but are not useful for studying immune-mediated responses to malignancy .As abdominal tumour development and its appearance in distant thoracal sentinel lymph nodes and regional spread of gynaecological or other malignancies –50 is noThe growth pattern of primary abdominal tumours in rodents  exhibited markedly similar features: peripheral ruptures of blood vessels near the surface of the primary tumour, shedding tumour cells into the abdominal cavity, crossing the stomata of the diaphragm and causing metastatic spread in thoracal, primarily parathymic lymph nodes (PTNs). Similarly, the intraperitoneal administration of Indian ink colloidal particles bypassed the distributing vascular route and tumour cells accumulated in PTNs. The thoracic duct drains into the systemic blood circulation at the angle of the left subclavian and internal jugular veins. At this point, the tumour spread becomes generalized and metastatic foci can develop wherever the circulation of blood is obstructed.18FDG-PET examinations. High differential absorption ratios  as”hot spots” as well as miniPET images of rats served as potential metastatic indicators for the early diagnosis of micrometastases before the upper thoracal lymph nodes would have been affected . Rodents, especially mice and rats, are the most popular animals for breast cancer research [It is forbidden to inject live cancer cells into patients since the highly unethical practice of injecting HeLa tumour cells into elderly patients . Basic aresearch .Connections among thoracic and mammary lymph nodes are poorly understood but could explain the metastatic differences not only between marsupials and mammals but also among mammalian species. The chain of human thoracic lymph nodes that continues in the axillary region suggests that in man and modified higher in mammals, these nodes are in a closer relationship than in marsupials Fig. a similarExperiments related to animal models , veterinMycotoxins are promising candidates but not yet applied as medicine. One of the noncerns is the high resistance of tumour cells toward chemotherapy  aggravatThe metastatic development of peritoneal and retroperitoneal primary tumours to parathymic lymph node metastasis reflects a general mechanism of tumour spread starting with the effusion of tumour cells of the primary tumour through the disrupted veins, crossing the diaphragm entering thoracal lymph nodes and potential progression into mammary lymph nodes.The data of Table The growth of perioneal and retroperitoneal tumors developing into parathymic lymph node metastasis reflects a general mechanism of tumour spread starting with the effusion of tumour cells of the primary tumour through the disrupted veins, crossing the diaphragm entering thoracal lymph nodes and potential progression into mammary lymph nodes.The human aspects of metastatic spread are based on animal experiments using tumour cell lines established from chemically induced rat tumours. The advantage of indirect xenografts created from cell lines, over original tumour explants without a cell line obtained from tumour-bearing rats was that direct tumour implantation into rodents could be avoided. The abdominal-thoracal route suggests a novel lymphatic connection between abdominal tumours and mammary lymph nodes and could provide the missing link between the abdominal tumours and breast cancer. Thoracal lymph carrying tumor cells can drain the opposite direction, when lymphatic blockade redirects tumour spread. The direct lymphatic connection between the diaphragm and mammary lymph nodes was published in Gray’s human anatomy . UnfortuBased on the structural analogy between mammalian lymphatic systems and functional similarities in tumour development and spread, it is reasonable to assume that tumour cells shredded the human abdomen cross the diaphragm and deposit in internal mammary lymph nodes (IMNs) underneath the chest wall, similarly to the accumulation of tumour cells in the parathymic lymph nodes (PTNs) in rats. Experimental tumour models helped to summarize the metastatic hypothesis and subdivide it into four phases. Abdominal primary tumour formation—phase 1. Growth of abdominal primary tumour, with necrotic cells inside the tumour (black) and reduced angiogenesis outward causing the release of tumour cells through the, disrupted blood vessels. Phase 2 passage of tumor cells through the diaphragm Fig. b Tumour www.oucom.ohiou.edu/dbms-witmer/downloads/2012-04-24_dashner_rpac-breastanatomy.pdf)Lymphatic vessels of the diaphragm follow the course of their corresponding vessels and terminate not only in the anterior and posterior mediastinal lymphatics but more importantly in the intercostal and internal mammary lymph nodes of the laboratory rat . This roImproved reproducibility, larger than murine body size, well-characterized and human-like physiology, made the rat tumour model indispensable for metastatic research. Another likely reason why human mammary lymph nodes in the development of breast cancer metastasis cannot be used is the hidden location of IMNs inside the thymic capsule. The rat metastatic model helped to recognize the spread of metastasis through the enlargement of PTNs, whereas the accumulation of tumour cells in murine and human PTNs enclaved in the thymic tissue remained undetectable. In addition to the findings of experimental metastasis, clinical data support the hypothesis that human breast cancer may originate from primary tumours.alias parathymic) lymph nodes, or spread via direct connections between the abdomen and the thorax in a lymphatic rather than a vascular manner. Anatomical evidence for the lymphatic connection between the diaphragm and mammary lymph nodes was provided [The tumour development in rodent models is supported by clinical and anatomical evidence suggesting that a considerable proportion of metastases in the breast may originate from internal mammary (provided .http://www.breastcancer.org/research-news/20110912). However, advances in radiation therapy technology make it unlikely that healthy nearby tissues would be exposed. Moreover, advances in diagnostic and surgical skills prove that IMN biopsy and removal can be performed safely without complications [Clinical investigations related to the lymphatic drainage of the breast reported the possibility of bidirectional movement to the axillary lymph nodes and drainage from the internal mammary lymph nodes , 95. Theications . Anotherications . Studiesications . These oications , 101.associations of breast lymph nodes exist between parasternal and thoracic lymphatic vessels,abdominal cancer cells tend to spread along lymphatic passages,main lymphatic drainage of the breast (75%) is via axillary nodes, but the remaining drainage (25%) is through parasternal nodes,unilateral lymphatic blockade may redirect tumour spread,www.oucom.ohiou.edu/dbms-witmer/downloads/2012-04-24_dashner_rpac-breastanatomy.pdf.lymph that is carrying cancer cells can also dinin to opposite te direction It is assumed that in mammals the tumour cells shed by primary tumours through the disrupted blood vessels of the primary tumour to the peritoneal and retroperitoneal cavities cross the diaphragm and are taken up by the thoracal lymph vessels, carried to and accumulated in the internal mammary lymph nodes. Several arguments have been listed supporting the idea that tumour-bearing lymph nodes among them mammary lymph nodes are not primary tumours, but metastases . This re18FDG-PET examinations. High DAR values as”hot spots” and miniPET images of rats served as potential metastatic indicators for the early diagnosis of micrometastases before other organs (e.g. mammary lymph nodes) would have been affected [The metastatic spread of tumour cells was supported by affected . Our vieaffected . Resultsaffected  among thaffected .The incidence of breast cancer, the most common malignancy in women increases with age, with the majority of patients diagnosed after menopause. In about 15–25% of cases, patients are premenopausal at the time of diagnosis of cancer, and about 7% are below the age of 40 [


Considering the applications of fluorescent probes and the information they provide, their brightness of fluorescence and photostability are of paramount importance. However, in the case of steady-state fluorescence spectroscopy and fluorescence microscopy, the amount of information can be increased by the application of multi-channel probes, via a multi-band fluorophore introduced in the probe molecule. In most cases, the use of such a multi-band (or multi-channel) fluorophore can also be combined with the concomitant introduction of one or several analyte receptors. Most often, the design of ratiometric probes with multi-band fluorescence emission are based on phenomena such as photoinduced intramolecular charge transfer (ICT) or excited state intramolecular proton transfer (ESIPT). Although ICT probes were up to recently the most popular, ESIPT probes and among them 3-hydroxyflavone derivatives, were shown to be the most productive. Several general problems were resolved by this family of probes, as for example the measurement of local dielectric constant, local H-bond accepting ability, water local concentration and ATP concentration in small volumes.H+, Mg2+and Ba2+ions, and thus to recognize these different cations.Incorporation of such multi-channel probes into lipid membranes allowed to measure the different membrane potentials and to detect cell apoptosis. Also, it enabled to recognize and characterize the rafts formation in different lipid bilayers and peculiar features of the charged membrane interface. Such probes are also able to provide a concentration-dependent fluorescence signals upon binding of The multi-channel probes are effective tools in the study of interactions of macromolecules such as peptides, proteins and nucleic acids. The most useful feature is that they inform simultaneously about several physical parameters, in this way giving a better insight in the investigated system. Thus, by comparing the reviewed probes with other modern fluorescent approaches, it can be concluded they are more informative and accurate tools.  At home and at work - everywhere we are now surrounded by multi-channel information transmitting devices, e.g. televisions, laptops, smartphones, etc., with numerous systems for organizing information flows. What are the prospects and trends on the organization of the smallest multi-channel devices - the fluorescent probes of a single molecule sizes - this problem will be discussed below.Fluorescence techniques are now widespread and effective tools for chemical and biological applications, especially in the study of objects at the molecular level Of course, intensiometric probes with the brightest fluorescence are the most popular now. However, on several reasons the structure of their fluorophore limits the possibilities of molecular design. The main problem is they are intensiometric, i.e. a single-channel and the least informative, reporting only the single studied parameter of the object. Nowadays, the increased sensitivity of devices for counting photons provides new opportunities for the use of fluorescent probes with increased informativeness, despite their somewhat lower brightness. This is especially important for the experiments in microheterogeneous solutions, e.g., in cells and tissues.Why is the informativeness of the probe so important and how best to organize the flow of information in steady-state spectroscopy? Every scientist will agree that the adequacy of a signal to event and its unambiguity, i.e., the fight against artifacts, is in the first place in experiment. The most vulnerable to generation of artifacts are intensiometric probes, in which a useful signal is transmitted through a single information channel - by changing the intensity of fluorescence. A change in intensity means a turning off/on a separate mechanism of fluorescence quenching during the probe - analyte interaction. Unfortunately, there are several quenching mechanisms known, and all of them may drive the probe fluorescence in solutions independently on probe - analyte interaction event. As a result, an experiment may imperceptibly enter the zone of obtaining artifacts. E.g., in microheterogeneous biosystems, a false signal is often may be caused by a changes in local viscosity of the probe surroundings or by changes in local concentration of water due to an unpredictable event.λ1I/λ2I, and its changes is based on the change of this ratio. That is what gave rise to the name of ratiometric method. Sometimes intensity ratio λ1I/λ2I may be transformed into band maximum position parameter, but they are not the same in complex cases. The creation and widespread application of ratiometric probes, where the fluorescence intensities at two wavelengths always presents an analytical signal with internal calibration can be considered as a first step on the multi-channel probes evolution.In order to prevent artifacts, it is important to simultaneously obtain data on several parameters of the molecular environment, such as composition of probe surrounding, including the presence of analyte, local viscosity, polarity, etc. In a steady-state fluorimetry it can be performed by creating of several information channels, such as signal intensity at several wavelengths. Here there is a requirement to divide the data on physical events by information channels. So that the influence of each physical parameter would be different part of content in each of the organized information channels. Currently, such direction is intensively developing in the aspect of two-channel, or ratiometric probes Ratiometric probes became popular after their fluorescent cores were synthesized, which was the first solvatofluorochromic dyes that change the color of fluorescence in the media of different polarity. In fact, there the change in color has always been an influence of several environmental parameters, such as intermolecular electric fields, the ability of atoms to donate and accept H-bonds, and viscosity of medium in part. The most popular representatives of this class, which have been widely used for a long time, include 5-(dimethylamino)naphthalene-1-sulfonate (DNS), tryptophan and PRODAN derivatives. They are used on practice until now, despite that recently a number of dyes with more pronounced solvatofluorochromism, bright red fluorescence and greater photostability appeared 1 state and to shift the emission band down towards higher wavelengths, in special cases with small changes of fluorescence brightness. This corresponds to the Intramolecular Charge Transfer (ICT) effect .,45.44,45N*/IT* of the probe fluorescence is in linear dependence with the Abraham's basicity parameter β of a solvent. A set of parameters composes the most popular scale of H-bond accepting ability  derivative of 3HF in order to detect Mgonitrile  49,50].,50.2+ an2+ and Ba2+cations. A new bathochromic band corresponding to complex 1b appears in the absorption spectrum when Mg2+concentration increases. This band is progressively undergoing a blue shift when the complex 1bc of stoichiometry 1:2 is forming. These variations can be explained by the sequential steps, where the first Mg2+ cation is bound by the hydroxy‑carbonyl chelator, in place of a proton, and the second one – by the macrocycle. Time-resolved spectroscopy of complex 1bc shows its partial ejection of Mg2+ from the crown-cycle in the excited state as a result of electron charge transfer from the nitrogen atom to the conjugated system piperasine-N’-ethylsulfonate (HEPES) buffer when labeling anionic egg yolk phosphatidylglycerol (EYPG) vesicles The mentioned above result was achieved due to the possibility of comparing the fluorescence intensities of the anionic form (460 nm) and the normal form (400 nm) in the excitation spectra. The shift of the band maximum of the anionic form to 460 nm in the lipid membrane compared to its position in water (430 nm) indicated its additional stabilization in the electrostatic field. Therefore, the use of three information channels of the fluorescence intensities at 400, 430 and 460 nm in the excitation spectrum and the analysis of the band shapes of the emission spectra made it possible to establish the presence of unique molecular complexes on the surface of the lipid membrane.The detailed study of HEPES-EYPG vesicles system by steady-state and time-resolved fluorescence spectroscopy allowed explaining this unusual phenomenon. It was suggested that the FME anion formation is a result of probe localization in the electrostatic field of the triple charged layer formed by EYPG-HEPES aggregates. The entering of FME probe into this layer is promoted by H-bond formation of the 3-OH group with basic nitrogen atom of HEPES molecule. In the absence of HEPES such an effect was not observed. The evidence of FME anion location in the electrostatic field is that the position of its maximum in the absorption and excitation spectra was anomalously shifted to the red in comparison with one in water solution. Thus, the specific structure of EYPG-HEPES surface results in partial dissociation of FME probe even at neutral pH values of bulk solution.Environment-sensitive labels present a powerful tool in the study of interaction of peptides with lipid membranes. The multi-channel fluorescent probes based on 3-hydroxyflavone fluorophore give the most detailed information about peptide surroundings in such experiments, as the analysis of their spectral parameters can differentiate the dielectric interactions from H-bonding ones l-lysine, they were covalently labeled at their N-terminus with 3HF probe NH*/IN*, as well as the position of the maximum of the N* band. The binding can be easily followed by the increase of fluorescence intensity  lipid vesicles and the lowest one for melittin in DOPC vesicles. A lower polarity means a deeper penetration of the peptide N-terminus into the lipid bilayer. This was confirmed by parallax quenching experiments using different location quenchers. The orientation of the label in giant unilamellar vesicles was estimated by fluorescence anisotropy of N* and T* forms emission in fluorescence microscopy experiments. It was evidenced that poly-l-lysine displays an orientation of the label orthogonal to the membrane surface, while melittin displays both parallel and orthogonal orientations. The work To study the membrane binding of three different peptides, melittin, magainin and poly-ntensity A,B. Melil-amino acid AFaa containing a 3HF fluorophore, which makes it possible to set the polarity and hydration of environment around a certain segment of the peptide. Such an exotic amino acid was introduced in two melittin peptide variants by solid phase synthesis instead of leucine-9 (L9) or tryptophan-19  increase in the fluorescence emission intensity and in the appearance of two emission bands. Such an observation is usual for 3HF probes embedded within the viscous hydrophobic lipid core of a bilayer. The label at position L9 shows the lowest IAlso should be stressed, the macrodipole of the alpha helix is poorly interferes with the charge transfer and ESIPT of the probe, since it is close to the orthogonal orientation relatively to the probe molecular dipole. Second, and the main issue, the probe dipole in any of orientations is surrounded by lipid molecules in membrane or by water in aqueous solutions. So, the charges of phosphocholine residue and the dipoles of fatty acid ester groups or water give main influence on the fluorescence of the AFaa probe.Fluorescence microscopy experiments with giant vesicles and live cells using a polarized light excitation show drastic differences in the orientation of AFaa label at L9 and W19 positions. This means that in cell membranes, as in lipid bilayers, melittin helix is preferentially oriented parallel to the membrane surface. Thus, the effective label construction and the combined utilization of several fluorescence parameters enabled to simultaneously obtain new and unique data about both peptide location and orientation in lipid membranes.A highly selective detection method of ATP anion in water was obtained by using the 4′-dimethylamino-3-hydroxyflavone , due mainly to the appearance of FME anionic form, as shown by the important red-shift t 60 nm, A,B in bo−6 to 5∙10−2 mol L − 1. The increased range of ATP detection was explained by the formation of two consecutive compleaxes probe-ATP with stoichiometry 1:1 and 1:2. The last work has shown that the 3-hydroxyflavone core is the scaffold that binds to ATP within its biological concentrations, and the substituents only partially change the affinity of the probe to ATP.In furtherance of these studies, an uracil-linked 3-hydroxyflavone The productive studies of lipid membranes with 3HF probes have stimulated the development of the labels for the study of peptide-nucleic acid interactions. To this aim, the peptide corresponding to the zinc finger domain of the human immunodeficiency virus HIV-1 nucleocapsid protein was synthesized, labeled by the N-terminus by 3HF probe and successfully applied to study the interaction with oligodeoxynucleotides (ODNs) and DNAs N*/IT* ratio were induced by the binding of ODNs of different length, sequence and strandedness, single-stranded (ss) or double-stranded (ds), to the labeled peptides  to the binding, in line with their higher sensitivity to the environment. Indeed, dramatic changes in the Ipeptides A. The le ds DNAs B. As a clα--amino-acid 3HCaa was synthesized on the basis of 2-furyl-3-hydroxychromone Further on, the elaborated method of quantitative evaluation of hydration N*/IT*, showing a stronger contact of A30 site of peptide with ΔP(-)PBS. In the case of W37 variant, on interaction with ODNs SL2, SL3 and ΔP(-)PBS, a strong change in the fluorescence spectrum was observed, with notably a large drop in the ratio IN*/IT*, which can be assigned to a decrease of the local hydration of the probe environment caused by the close distance to RNA segment . These results can be rationalized by a larger distance between A30 residue and the nucleic bases within the complexes an consequently a higher exposition to water. These data correlate well with the NMR data for the NC(11–55) peptide and for the whole NC sequence PBS sequence additionally results in the decrease of the intensity ratio Isequence B,C.A parameter can be calculated from the calibration procedure A parameter drops to 0.32–0.41 for W37 variant, but only to 0.49 −0.54 for the A30 variant, to be compared to 0.57 for the free labels.In both cases, the water access Wl-amino acid based on a 3HC probe, incorporated at different positions by peptide synthesis into a peptide, opens the possibility not only to quantitatively characterize the interaction of the peptide segment with other peptide parts or with nucleic acids, but also to determine the level of water access at the interaction site. Such higher informational content directly results from a clever utilization of this family of multi-channel probes, as compared to more classical probes  was synthesized, incorporated into NC(11–55) peptide at the same A and W sites and studied in an experiment on binding with oligodeoxynucleotides (ODNs) and lipid vesicles (LUVs) In the following work a new The work on study of the single-stranded and double-stranded ODN complexes 1 site does not significantly reduce the antibacterial activity, although it slightly changes the conformation of the peptide in model solutions. The equilibrium between two conformations of the peptide in lipid membranes, micelles, trifluoroethanol and buffer solution was suggested by the data on the position and intensity of bands in the fluorescence and absorption spectra. In this work the maximal number of information channels created by the probe in the steady-state spectra was applied. The shape and band maximum position in the fluorescence and absorption spectra were analyzed. For example, for all tested peptide variants due to the presence of three cationic groups of arginine, a significant difference in the conformations of the peptide was registered according to the position of the maximum in absorption and fluorescence excitation spectra obtained in lipid membranes (360 nm), water (370 nm), ethanol (440 nm) and dimethylformamide (450 nm).Labeling biomolecules with fluorescent probes is an established tool for biophysical studies. However, it remains underused for small peptides, as there is a serious threat in such case to change affinity and selectivity of the peptide interaction with its target in the object. In a recent work, the fluorescent amino acid probe 3HCaa was incorporated into a known antimicrobial peptide, cyclo[RRRWFW] (cWFW), in place of aromatic residues Comparing the results obtained with other probes on cWFW peptide modification, 3HCaa can be considered the first adequate label for the study of small peptides. This feature allowed to get insights into conformational equilibria of the labeled peptides localized in cells, and assess the polarity and hydration of the local environment around the label by confocal fluorescence microscopy. It was found that the labeled peptides efficiently penetrated cancerous cells and localized mainly in lipid-containing and/or other nonpolar subcellular compartments. In the zebrafish embryo, the peptides remained in the bloodstream upon injection into the cardinal vein, presumably adhering to lipoproteins and/or microvesicles. They did not diffuse into any tissue to a significant extent during the first 3 h after administration. This study demonstrated the advantages of fluorescent labeling by multi-channel probes to elucidate the sites and mechanism of action of peptides in organism.Fluorescence of a dye in solution can be too complex a phenomenon to fully interpret the nature of its fluorescence. The Jablonsky diagram  only parTwo phenomena are frequently used in the design of ratiometric probes with multi-band fluorescence, namely photoinduced intramolecular charge transfer (ICT) and excited state proton transfer (ESIPT). Despite ICT was up to now the most popular one, the introduction of ESIPT, more particularly for 3-hydroxyflavones and 3-hydroxyquinolones probes, is presently becoming very attractive. This is due to additional channels of information created by these probes by changing the intensity and position of each of the bands in the fluorescence excitation and emission spectra. Each channel can be separately tuned either by the structure of the fluorophore or the presence of different analyte receptors and transmit information about several physical parameters of probes location. This strategy led to new achievements, including the measurement of local dielectric constant in the probe environment, local H-bond accepting ability, local concentration of water and concentration of ATP in aqueous solutions, all tasks which were quite difficult to achieve with ordinary intensiometric probes.In most cases, the main informational channel, which is usually the intensity ratio of the two emission bands is used as the main tool for the observation of the process under study, while the other channels  are more often used to control other possible processes in experiments.Such multi-channel probes were used to noncovalently label lipid membranes, allowing for example to measure dipole and transmembrane potentials and to detect cell apoptosis. They allow also deciphering the structure of raft membranes and also some particular effects concerning the influence of HEPES buffer molecules on the interfacial behavior of phosphatidylglycerol vesicles.H+, Mg2+and Ba2+ cations, giving the opportunity to selectively recognize these cations of different radii by a single probe. Also these probes allow sensing the pH in a wide range.In the field of cation sensing, such multi-channels probes are able to distinguish between different fluorescence signals upon binding with Multi-channel probes are effective tools in the study of macromolecule interactions. The most useful peculiarity is that they inform simultaneously about several physical parameters of the environment, such providing a better insight about the studied system. As an example, the total fluorescence intensity of such probes could report about local viscosity, the N*/T* band intensity ratio – about local hydration, and the position of N* band – about local polarity, while the polarization of fluorescence could report more about orientation of the probe or even the whole macromolecule. Labeling of peptides by amino acid-mimicking multi-channel probes, via peptide synthesis, opens a new route to study peptide location, and level of peptide site interaction with other biopolymers . The orientation of an α-helix peptide relatively to a cell membrane surface can be also determined by this way.Thus, comparing multi-channel probes presented in this review with other more classical fluorescent probes, it can be concluded that they are more informative and more accurate tools.Since proton is the lightest nucleus of an atom and its migration in a molecule leads to striking changes in electronic structure together with significant and fastest changes in fluorescence properties of the molecule in time scale, perhaps that is why the phenomenon of ESIPT has become the most convenient in the creation of multi-channel fluorescent probes. A logical step to increase the number of information channels from the probe was the development of dyes with two proton transfer systems, both same in structure or different, located in close proximity or at different poles of the molecule Another approach to increase the number of information channels of the probe was the combination of ESIPT and excitation energy transfer phenomena ,24. In sAfter all, in the design of the probe not only ESIPT can be exploited, but several types of chemical reactions. For example, nucleophilic addition or redox type ones. There are already many examples ratiometric (two-channel) probes of such kind design By this letter I declare that I have no conflict of interest and no competing interests such as those usually defined for manuscripts of articles or others that might be perceived to influence the results and discussion reported in the paper named as Multi-Parametric Sensing by Multi-Channel Molecular Fluorescent Probes Based on Excited State Intramolecular Proton Transfer and Charge Transfer Processes.