Three hits based on 1,2,3-triazole moiety represent the periphery of the recently reported potent inhibitors of hCA II, IX and XII which were efficacious in vivo. Such a re-discovery of suitable BSA periphery essentially validates the new fragment-based approach to the discovery of future CAIs. Structures of other validated fragment hits are reported.The differential scanning fluorimetry (DSF) screening of 5.692 fragments in combination with benzenesulfonamide (BSA) against bovine carbonic anhydrase (  It is, therefore, denoted as a zinc-binding group (ZBG). It is, however, the CAI molecule’s periphery that determines the potency and selectivity. This is illustrated by the evolution of the weak and non-selective CA inhibitor benzenesulfonamide (BSA)1–4 1–4 8–11.dK 0.1…1.0 mM) affinity. Considering the low molecular weight of fragments, the ligand efficiencyDSF is a versatile biophysical platform for fragment-based drug discoveryWe reasoned that if the low-affinity BSA is screened in combinations with various fragments, the combinations that cause greater thermal shift than BSA or fragment in question alone could signify suitable BSA periphery for the design of novel CAs. In other words, this could provide a basis for the discovery of fragments that bind to CA in cooperative fashion with BSA and thus can serve as candidates for subsequent chemical linking  to the BSA motif eventually leading to potent CAIs .bCA) as a model CA enzymemT) of BSA and thus could be considered cooperative binders while some of the fragments thus discovered represent hitherto undescribed periphery for BSA-based CAs and will be pursued in further studies, one set of fragments corresponded to the recently described series of potent BSA-based CAIs. We consider such a rediscovery an important fact that validates the fragment-based drug discovery approach proposed above. Herein, we present this significant result in detail.We tested this strategy using a set of 5692 diverse fragments from the Enamine, Ltd. Screening CollectionSupplemental Material) for the DSF screening were selected by substructure search and obtained directly from the Enamine screening collection1H NMR spectroscopy prior to biochemical testing in CA inhibition assay. The majority of these compounds (3344) had molecular weight lower than 200 and were characterised by low polarity bCA binder at 20 µM concentration.Thermal shift assay was carried out using ViiA™ 7 Real-Time PCR System equipped with 384-well block . The TSA procedure was adopted from the literature2PO4/Na2HPO4, pH 7.0, 10 mM NaCl. The total volume of the reaction mixture per well was 10 µL. Carbonic anhydrase (Sigma Aldrich Cat# C3934) in 300 µg/mL concentration was pre-mixed with environment-sensitive SYPRO orange dye  at final concentration in the reaction of 10x, with regard to the stock concentration stated by the vendor. The mixtures were pre-incubated for 1 h at 4 °C with 20 µM concentrations of the compounds , placed into MicroAmp® Optical 384-Well Reaction Plate . The reaction mixture was kept at room temperature for 15 min to ensure full protein-compound interactions. The temperature was raised at 1.6 °C/s rate to 40 °C without signal reading. Starting from 40 °C up to 85 °C the heating rate was set to 0.05 °C/s with constant fluorescence reading, using 470/623 nm filter set. The raw data of dye fluorescence intensity change upon melting of the protein were obtained from the instrument ViiA 7 RUO software. Further data processing and visualisation was performed by custom-made Microsoft Excel scripts. The peak of the first derivative for the fluorescence curve was used to define melting temperature mT. mT for DMSO control wells, having only protein, dye and 1% DMSO was used as a oT to determine melting temperature shifts (ΔmT). All measurements were performed in quadruplicates.For the fragment screening, the test reactions were set up in the following buffer: 10 mM NaHEach fragment was screened either alone (50 µM) or, in the same concentration, in the presence of BSA (50 µM). Compounds that displayed significant (>0.5 °C) change in the thermal shift produced by BSA were subsequently tested, in quadruplicates, at three different concentrations .bCA melting temperature (ΔmT) which was dose dependent (bCA tertiary structure on BSA binding.Benzenesulfonamide (BSA) alone produced a noticeable positive thermal shift of mT value of BSA was significantly lower than the one observed for AZ, a potent CA inhibitor, at 20 µM concentration (>5.0 °C)As expected, the ΔSupplementary Material). The vast majority of these hits will be employed in subsequent studies either as the basis for their evolution into potent CA inhibitors on their own . However, in terms of the cooperative effect with BSA, three fragments in particular (5–7) attracted our attention as they all contain a common 1,2,3-triazole core , when tested as such against bCA by DSF, turned out to be negative thermal shifters, i.e. they decreased the protein melting temperature . This is indicative of their binding being a destabilising factor to the tertiary structure of the protein (vide supra (5–7 likely represent suitable prototypes for the development of BSA into potent CA inhibitors via addition of periphery groups to the relevant positions of the benzene ring.These three hits ( protein 23. Howev5–7 is significant in light of the recently reported8–9 of cytosolic hCA II (glaucoma target) as well as membrane-bound hCA IX and XII (cancer targets) that are based on benzenesulfonamide decorated with a 1,2,3-triazole moiety attached via a flexible linker  delivered >100 hits that either caused, on their own, a significant thermal shift  in the protein melting temperature or significantly influenced the thermal shift observed for BSA alone. Three such hits were of particular interest as they most significantly altered the thermal shift of BSA and are structurally related to each other and to the periphery of the recently reported series of potent hCA inhibitors which were efficacious in vivo. The findings reported in this Communication essentially validate the novel fragment approach to the discovery of new inhibitors of carbonic anhydrase. This approach is expected to eventually alleviate the need to screen larger libraries of compounds to identify potent hits. Our focus is currently on extending this approach to other fragments containing a primary sulphonamide moiety. The results of these studies will be reported in due course.The differential scanning fluorimetry screening of 5.692 fragments in combination with benzenesulfonamide (BSA) against bovine carbonic anhydrase (Click here for additional data file.


Cigarette smoke inhalation exposes the respiratory system to thousands of potentially toxic substances and causes chronic obstructive pulmonary disease (COPD). COPD is characterized by cycles of inflammation and infection with a dysregulated immune response contributing to disease progression. While smoking cessation can slow the damage in COPD, lung immunity remains impaired. Alveolar macrophages (AMΦ) are innate immune cells strategically poised at the interface between lungs, respiratory pathogens, and environmental toxins including cigarette smoke. We studied the effects of cigarette smoke on model THP-1 and peripheral blood monocyte derived macrophages, and discovered a marked inhibition of bacterial phagocytosis which was replicated in primary human AMΦ. Cigarette smoke decreased AMΦ cystic fibrosis transmembrane conductance regulator (CFTR) expression, previously shown to be integral to phagocytosis. In contrast to cystic fibrosis macrophages, smoke-exposed THP-1 and AMΦ failed to augment phagocytosis in the presence of CFTR modulators. Cigarette smoke also inhibited THP-1 and AMΦ mitochondrial respiration while inducing glycolysis and reactive oxygen species. These effects were mitigated by the free radical scavenger N-acetylcysteine, which also reverted phagocytosis to baseline levels. Collectively these results implicate metabolic dysfunction as a key factor in the toxicity of cigarette smoke to AMΦ, and illuminate avenues of potential intervention. Among those consequences is chronic obstructive pulmonary disease (COPD), which is projected to soon be the third leading cause of death worldwide5. As the principal innate immune cell in the healthy lung, alveolar macrophages (AMΦ) are implicated in dysregulated inflammation and cycles of recurrent and chronic infections of COPD, yet the direct toxicity of cigarette smoke to ΑΜΦ remains incompletely understood6. AMΦ are responsible for clearing pathogens, particulate matter, and cellular debris from the lung, and any impairment in this process is likely to have deleterious physiologic consequences7. For example, poor phagocytosis is known to negatively correlate with lung function in COPD8. Conversely, rescuing diseased AMΦ function may prove of therapeutic benefit; azithromycin has been shown to augment phagocytosis of apoptotic bronchial epithelial cells  in COPD AMΦ10. In addition to impaired efferocytosis12, COPD AMΦ are known to be defective in phagocytosis of bacteria16, however data on the acute effects of cigarette smoke on AMΦ are lacking.Cigarette smoking infuses the respiratory tract with toxins, causing abundant, long-lasting consequences even after smoking cessation17. CFTR dysfunction has been demonstrated in bronchial epithelial cells from COPD patients, presumably contributing to mucous hyperviscosity and ciliary stasis with subsequent mucous plugging, impaction, and chronic infections22. The CFTR potentiator ivacaftor  is FDA approved as monotherapy for CF patients with CFTR gating mutations24, and is a component of combination therapies for patients with trafficking mutations29. As such its use has significantly improved outcomes for CF patients30. It has also shown promise in reversing cigarette smoke-induced CFTR dysfunction in vitro and is currently in clinical trials for patients with chronic bronchitis32.An emerging area of research into COPD pathophysiology relates to the role of the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride and bicarbonate channel which is mutated in cystic fibrosis (CF)34 in a manner that is reversible by the CFTR corrector lumacaftor35, raising the possibility that CFTR activity is required for optimal function of COPD MΦ as well. Herein we investigate the effects of cigarette smoke on AMΦ phagocytosis of Pseudomonas aeruginosa, CFTR expression, and the potential salutary effects of CFTR modulating drugs.CFTR dysfunction has been shown to impair phagocytosis in CF MΦ39. Inflammatory activation of MΦ causes them to shunt glucose towards glycolysis in lieu of mitochondrial oxidation, and conversely, modulating metabolism impacts inflammatory state40. Availability of glucose has been shown to modulate phagocytosis as well as glycolytic rates in an in vitro diabetes model41. Here we investigate how cigarette smoke impacts MΦ metabolism and reactive oxygen species (ROS) generation, and probe the activity of the free-radical scavenger N-acetylcysteine (NAC) in rescuing smoke-induced dysfunction.MΦ phagocytosis is also understood to be intertwined with metabolism and reactive oxygen speciesP. aeruginosa utilizing a gentamicin protection assay. We chose a 20 min incubation with bacteria, which was previously demonstrated to be within the linear phase of bacterial ingestion35. Using THP-1 cells differentiated into MΦ by addition of the protein kinase C agonist phorbol 12-myristate 13-acetate (PMA), we found a large decrease in P. aeruginosa uptake after a 20 minute pretreatment with 10% CSE  on phagocytosis of CSE Fig. , a conceCSE Fig. . In primCSE Fig. .Figure 115, there are no published reports on the acute effects of CSE on bacterial phagocytosis in healthy human AMΦ. Here we found that CSE induces a dose-dependent decrement in phagocytosis in MΦ from all three right lung lobes as well as pooled MΦ with no significant differences in CSE effect between lobes  on phagocytosis. RLL ΑΜΦ had lower baseline phagocytosis levels when compared with RUL (p < 0.05) or RML (p < 0.001) however there was no significant difference in their respective sensitivities to CSE.While there are reports describing decreased phagocytosis in AMΦ purified from patients with COPD and “healthy” smokers relative to non-smokersbes Fig. . While tbes Fig.  and MDM bes Fig. . Analyzi42 as well as reported decrements in CFTR expression and function in smoke-exposed epithelial cells45, we determined the effects of CSE on CFTR expression in primary AMΦ. We exposed AMΦ to 5% CSE as this induced an intermediate inhibition of phagocytosis and was therefore felt to be an appropriate concentration for downstream experiments. We reasoned that if CFTR was relevant to phagocytosis under the short-term conditions in Fig. Given the importance of CFTR for phagocytosis35, we sought to determine the effects of CFTR modulators on CSE-induced defects. Using previously published concentrations of ivacaftor (30 nM) and lumacaftor (3 μM) which were in turn derived from serum levels found in CF subjects35, we pre-treated THP-1 derived MΦ  with an extracellular flux analyzer after CSE injection. We performed a mitochondrial stress assay which allows for the quantitation of basal respiration, ATP-linked respiration, maximal respiration, proton leak and non-mitochondrial respiration48. Figure Given the lack of effect of ivacaftor and lumacaftor on the phagocytosis defect, we sought to understand what other pathways besides CFTR dysfunction were involved. MΦ metabolism has been demonstrated to be integral to function49, we tested the presence of free radicals using the probe 2′,7′-dichlorofluorescin diacetate (DCF-DA), which is a ROS-activated fluorophore. There was a dose-dependent increase in the production of ROS by THP-1 MΦ in the presence of CSE  for further investigation. Figures 51 which is a known inducer of oxidative stress, however we saw similar effects in right upper lobe and right lower lobe AΜΦ.Mitochondrial dysfunction and ROS can form a feedback loop whereby mitochondrial ROS can further inhibit mitochondrial function−9) dose-dependent effect of hydrogen peroxide. Further implicating ROS in this process, NAC reversed the phagocytosis defect of either hydrogen peroxide or CSE  concentrations needed to produce effects in vitro, which could be difficult to attain in vivo.NAC has had mixed results as a treatment for COPD; while there are some data that it can decrease exacerbations at high doses in a cohort of patients who are subject to frequent exacerbationsex vivo system presented herein has strengths in that it allows us to evaluate the acute effects of CSE, it is also limited by that acuity. Administration of CSE is quite different from the chronic exposure to which smokers’ lungs are subject, and it will be interesting to see the relative contributions of CFTR and oxidative stress in smoker and COPD AMΦ. These two pathways are not mutually exclusive as CFTR itself is known to be a glutathione transporter59, and oxidative stress is exacerbated by CFTR dysfunction impairing the normal glutathione response60. Oxidative stress can then further decrease CFTR levels61, forming a vicious cycle. Experiments to illuminate these possibilities are ongoing in our laboratory. The source and identity of the elevated ROS will likewise be informative.While the experimental in vitro. Further studies will extend this work into primary AMΦ isolated from current smokers.In summary, we find that cigarette smoke acutely inhibits bacterial phagocytosis as well as mitochondrial function in human AMΦ. While CSE also decreases CFTR levels, CFTR modulators ivacaftor and lumacaftor are unable to rescue CSE defects. Meanwhile, NAC partially reverses the dysfunction, consistent with increased oxidative stress seen in the presence of CSE. This provides further basic mechanistic rationale for the use of antioxidants in COPD, somewhat tempered by the high concentrations needed to see an effect 51.This study was approved by the Committee for the Protection of Human Subjects at the Geisel School of Medicine at Dartmouth (CPHS protocol #22781), and all procedures were performed in accordance with relevant guidelines and regulations. Healthy subjects  were enrolled if they were between 18 and 55 years of age, non-smokers, and had no underlying pulmonary disorder or significant comorbidity. Informed consent was obtained from all participants for participation in research, phlebotomy, and for bronchoscopy individually. Phlebotomy (100 mL whole blood for monocyte isolation) was performed at the time of peripheral IV insertion. Next, after local anesthesia to the posterior oropharynx, with or without systemic sedation per patient preference, a flexible bronchoscope was passed through the mouth and vocal cords until wedged into sub-segmental bronchi of the right lung. Five lavages each of 20 cc 0.9% saline and 10 cc air were performed in the right upper lobe, right lower lobe, and right middle lobe sequentially. Bronchoalveolar lavage (BAL) samples from individual lobes were obtained and alveolar macrophages purified as previously describedSources of reagents are available in the online Supplementary Information.62. One cigarette was lit and attached to 5 cm plastic tubing and placed on the end of a 60 mL syringe containing 10 mL of media. Smoke was drawn into the chamber of the syringe until 50 mL total volume was obtained, then agitated until the smoke cleared. This process was repeated until the cigarette was consumed, which was defined as 100% CSE.CSE was purified according to protocol of Blue and Janoff as described by others35 was used to quantify phagocytosis. Specific details are available in the Supplementary Information.A modified gentamicin protection assayPrimary alveolar macrophages were plated overnight, then treated with 5% CSE for one hour and stained for CFTR, followed by counterstaining with Hoechst to reveal nuclei. Specific details are available in the Supplementary Information.Macrophages were plated onto Seahorse assay plates obtained from the manufacturer at 50,000/well. Assay was run according to manufacturer’s protocol for mitostress assay and glycolytic rate assay, respectively. CSE was added to port A to be injected prior to kit compounds in ports B through D respectively. Reagent concentrations and buffer composition are in the Supplementary Information.2O2 (250 μM). AMΦ were cultured in the presence of 50 μM β-mercaptoethanol to reduce basal ROS. Cells were incubated in a Tecan plate reader at 37 °C and fluorescence readings  were taken every 5 min for one hour then every 30 min for 23 hrs. Readings of identically treated wells without dye were subtracted to normalize for fluorescence from media.Macrophages were loaded with 10 μM 2′,7′-dichlorofluorescin diacetate (DCF-DA) for 30 min, washed once with PBS and treated with vehicle or NAC 4 mM for 30 min, followed by the addition of CSE or HAll graphs were prepared with GraphPad Prism 7.0 . Linear models were run in R as indicated in text and figure legends. ANOVA and Mann-Whitney tests were performed in Prism. All p values are two-tailed and p < 0.05 was interpreted to be statistically significant. All data generated or analyzed during this study are included in this published article (and its Supplementary Information files).Online supplement


Health professionals are to play an essential and effective role in tobacco control. Toombak is a popular form of smokeless tobacco, locally made and used among Sudanese. It is associated with oral and systemic health hazards, particularly oral cancer. This study was set out to assess health professions students’ perception of their status as future role models for their patients and the public regarding Toombak use cessation and to explore socio-cognitive covariates of perceived role model status.A cross-sectional study was conducted in 2013–2014 using a self-administered questionnaire among health professions students enrolled in Medicine, Pharmacy, Dentistry and Nursing colleges in Khartoum state, Sudan. A total of 1530 participants were recruited from colleges using a cluster stratified sampling procedure.The majority of the participants (88.2%) were 19–22 year old, females showed predominance (66.1%). Most of the participants (69.7%) scored good knowledge of Toombak-related therapies. The majority (97%) had a positive attitude towards banning Toombak use in public. Half of the participants (50.4%) confirmed availability of Toombak control policy in their college, while 66% reported receiving training in Toombak cessation. Only 9.2% of the participants reported that they have ever used Toombak; among them, 69.5% were current daily users. Less than half (47.8%) reported a positive perception of their status as a role model. Logistic regression model revealed that tobacco cessation training, availability of college policy and positive attitudes towards banning Toombak use in public were strong predictors of with a positive perception of role model status.Imposing Toombak control policies within colleges combined with formal training in cessation methods, would reinforce the positive perception of health professions students as a role model, particularly the students themselves seem to be ready for such systematic training. Disability- Adjusted Life Years) and over a quarter of a million deaths occur each year [The tobacco epidemic is one of the main public health problems the world has ever met . It is each year . Cessatiach year .Nicotiana rustica. Toombak has the highest level of nicotine and carcinogens such as tobacco-specific nitrosamines (TSNAs) and nicotine-derived nitrosamine ketone (NNK) levels which are 50–100 times greater than the maximum concentration found among other tobacco products in other parts of the world [Toombak is a form of SLT locally made and consumed in Sudan. It is used as an oral snuff and is made from finely grounded leaves of he world . It is ehe world –9. In Suhe world World Health Organization (WHO) defined health professionals to include “physicians, nurses, midwives, dentists, psychologists, psychiatrists, pharmacists, chiropractors and other health-related professionals” . Health Health professionals’ role model status is determined by the fact that they are recognised to be health experts and the most knowledgeable than patients and society . They arPerceptions of health professionals of their status as a role model for their patients in tobacco cessation and prevention may be reflected by their attitudes towards tobacco use, and by their advice to patients and the public about tobacco cessation and prevention , 19. ThiWHO support the inclusion of tobacco cessation programs in health-education and services, and that all health providers should become advocates for tobacco control . WorldwiWhereas previous research has explored HPS’ attitudes towards and use of tobacco, few studies have considered the use of Toombak and HPS perception of themselves as role models for their patients and public in Toombak cessation activities. Thus, more information is needed to focus on the use of Toombak, as it is used by almost 38% of the Sudanese people . This stA cross-sectional study was conducted using a self-administered questionnaire among HPS enrolled in Medicine, Pharmacy, Dentistry and Nursing colleges in Khartoum state, Sudan, during 2013–2014.In Khartoum state, a total of about 20,000 health professions students were enrolled in 55 colleges during the study period. There are 18 colleges offering medical degree, 15 offering pharmacy degree, 13 offering dental degree, and nine offering nursing degree . Using cThe questionnaire and study information sheet were administered in English as it is the language used for teaching among the HPS in Sudan. A brief introduction to the research objectives was provided by the researcher. No personal identifying information was obtained and therefore the individual responses were kept anonymous.Demographic characteristics (3 items), knowledge of Toombak-related therapies (2 items), Toombak cessation training information and college policy (6 items), perceived role of health professions students (4 items) and attitudes toward the Toombak control (3 items), and Toombak use (2 items). A pilot study was carried out among 150 HPS (included in the study). Accordingly, no further major changes were made to the questionnaire.The questionnaire used in this study was based on the Global Health Professional Students Survey (GHPSS) questionnaire. The GHPSS is a college-based survey of 3rd-year students pursuing degrees in Medicine, Pharmacy, Dentistry, Nursing and other health professions, that has been developed by the WHO, the Centres for Disease Control and Prevention  in 2005 . The finKnowledge of Toombak cessation therapies was measured by two questions “have you ever heard of using nicotine replacement therapies in tobacco”; “have you ever heard of using antidepressants in tobacco cessation programs”; using response options (1) “yes”; (2) “no”; (3) “I don’t know”. The original categories were recoded into (0) “no” ; (1) “yes” . A sum variable labelled “knowledge of Toombak cessation therapy” (r (0.38), p <0.01) was constructed from the two questions (0–2). This sum was recoded into (0) no knowledge ; (1) good knowledge .Toombak cessation training at college was measured by five questions; “during your college training, have you ever received any formal training in Toombak cessation approaches to use with patients”; “did you taught in any of your classes about the health hazards of Toombak”; “did you discuss in any of your classes the reasons why people use Toombak”; “did you learn that it is important to record Toombak use history as part of a patient’s general medical history”; “did you learn that it is important to provide educational materials to support Toombak cessation to patients who want to quit Toombak”. Using response options (1) “yes”; (2) “no”; (3) “I don’t know”; the original categories were recoded into (0) no training ; (1) yes, there is training . A sum variable labelled “Toombak cessation training at college” (Cronbach’s α = 0.94) was constructed from the five questions (0–5) and recoded into (0) no training (includes 0); (1) yes, there is training .availability of college policy; “does your school have an official policy banning Toombak use in school buildings and clinics”; using response options (1) “yes, for school buildings only”; (2) “yes, for clinics only”; (3) “yes, for both school buildings and clinics”; (4) “no official policy”; the original categories were recoded into (0) no ; (1) yes .One question measured attitude towards banning Toombak use in public; “should Toombak sales to adolescents (persons younger than 18 years old) be banned”; “should Toombak be banned in restaurants” and “should Toombak be banned in parties”; using response options (1) “yes”; (2) “no”; (3) “I don’t know”; the original categories were recoded into (0) “negative attitude” ; (1) “positive attitude” . A sum variable “attitude towards banning Toombak use in public” (Cronbach’s α = 0.88) was constructed from the three question (0–3) and recoded into (0) positive attitude ; (1) negative attitude .Three questions measured Ever users of Toombak mean having used Toombak at least once or twice in their life. Current users of Toombak were defined as having used Toombak at least once in the last 30 days either daily or near-daily [ar-daily .Ever use of Toombak was measured by one question; “have you ever tried or experimented with Toombak, even one or two snuffs” using response options (1) “yes”; (2) “no”.Health professions students’ perception of their status as future role models in Toombak cessation was measured by four questions; “do health professionals serve as “role models” for their patients and the public”; “should health professionals routinely advise their patients to quit Toombak”; “do health professionals have a role in giving advice or information about Toombak cessation to patients”; “are patients’ chances of quitting Toombak increase if a health professional advises him or her to quit”. Using response options (1) “yes”; (2) “no”; (3) “I don’t know”; the original categories were recoded into (0) negative perception ; (1) positive perception . A sum variable “perceived role model” (Cronbach’s α = 0.90) was constructed from the four question (0–4) and recoded into (0) negative perception ; (1) positive perception  [ories 4) .2) to the level of explanation in the variance of the model.Data were analysed using the Statistical Package for the Social Science, version 20 (IBM SPSS Statistics). Descriptive analyses were performed using frequencies and percentages. Cross-tabulation using Chi-square, Odds Ratio (OR), and Confidence Interval (CI) assessed bivariate relationships between the dependent variable (perceived role model) and all independent variables. Multiple variable analysis was conducted using logistic regression with those independent variables that showed significant associations with the outcome variable in the bivariate analysis. Estimates were presented as OR and 95% CI, in addition to Nagelkerke’s , Nursing (14.8%) and Dentistry (14.2%). The majority of the students (88.2%) were 19–22 years. The gender distribution showed a predominance of females (66.1%), and this was the trend in all subgroups except for medical students .Most of the participants (69.7%) scored good knowledge of Toombak-related therapies. Almost all of the participants (97%) reported positive attitude towards banning Toombak use in public. Half of the participants (50.4%) confirmed availability of Toombak control policy in their college, while 66% reported receiving training in Toombak cessation.Only 9.2% of the participants reported that they have ever used Toombak; among them, 69.5% were current daily Toombak users. The highest frequency of Toombak use was among Medicine students (53.6%) and the lowest was among Nursing students (4.3%).Almost half of the participants (47.8%) reported a positive perception of their status as a role model for their patients and the public regarding Toombak cessation. Among Nursing and Medicine students, Pharmacy and Dentistry students were 52.9%, 53%, 40.3% and 42.9%, respectively. Furthermore, most of the students (82.4%) believed that there is an increase in the chances of quitting tobacco if a health professional advises his/her patients to quit. The majority of the participants (75.6%) perceived that health professionals have a role in giving advice about Toombak cessation to their patients .As shown in 2 of 0.022, explaining 2.2% of the variance perceived role model status. The final model indicated that tobacco cessation training, availability of college policy and attitudes towards banning Toombak use in public were significantly associated with a positive perception of role model status. As shown in Perceived status as a role model was regressed on age, gender, attitude towards banning Toombak use in public, training on tobacco cessation and availability of college policy. This model revealed a Nagelkerke’s RThis study was carried out to investigate Sudanese HPS’ perceived status as a role model in Toombak control and its possible covariates. It revealed that less than half of the participants reported a positive perception regarding HPS’ role model status in Toombak cessation. Moreover, perceived role model status was associated with their attitudes towards banning Toombak use in public, the availability of training and college policy about tobacco control.The findings of this study showed a lower percentage of students confirmed a positive perception of their role model status in Toombak cessation than a recent study by Sreeramareddy , in whicOne of the important covariates of a positive perception of role model status in this study is the formal training included in the curriculum of HPS. A total of 44% of the HPS reported no formal training in tobacco cessation in the curriculum. This finding is in accordance with several studies confirming that the lack of enough formal training is a widespread problem , 37–39. g. In Greece, the pharmacy students were reported the least trained group [This study reported disparities among the HPS formal training of Toombak cessation, as a higher percentage of Nursing students reported to have formal training than their counterpart in other colleges. This is consistent with 2007 GHSPS in Sudan . The dised group . Therefoed group .This study reporetd that students who did agree with having a positive attitude towards banning Toombak use in public places, were most likely to have a positive perception of their status as a role model. Studies have reported attitudes towards tobacco ban, associated with their knowledge of harmful effects of tobacco use, and their own status of using tobacco were important factors in shaping their perception as a role model . In addiThis study has various limitations; firstly, it focuses on third-year HPS, which has not had considerable contact with patients as the clinical education usually, starts during the third year of their professional education. Their perceptions might change over time. Secondly, the data are a self-reported, which can be subject to problems such as social desirability and recall bias. To cope with such potential bias, all measures to ensure the anonymity of the respondents and to keep the study anonymous was assured. The major strengths of this study are the use of a large, broadly representative sample of students from institutes in Khartoum, Sudan. The reliability of the questionnaire aligns with the GHPSS used before in Sudan. consistent evidence indicates that results obtained from such data are reliable . In addiThe findings of this study may have direct implications for health education and the involvement of Sudanese health professions in the promotion of tobacco control. This study reports the influence of various factors on positive perceptions of HP students regarding their status as a role model for tobacco control in Sudan. Further research will be needed to confirm these findings, and determine whether these findings can be used to advocate the need to mould HPS perceptions.Imposed Toombak control policies within colleges combined with formal training in cessation methods, would reinforce the positive perception of HPS towards their status as role models to their patients and public towards Toombak control, particularly the students themselves seem to be ready for such systematic training. Thus, health colleges in Sudan should be encouraged to incorporate Toombak-related issues in curricula, and to design and implement training programs for HPS that will enable them to develop the basic skills and capacity to provide effective Toombak cessation methods to their future patients.S1 File(SAV)Click here for additional data file.


Background and study aims Endoscopic surveillance for Barrett’s esophagus (BE) is limited by long procedure times and sampling error. Near-infrared (NIR) fluorescence imaging minimizes tissue autofluorescence and optical scattering. We assessed the feasibility of a topically applied NIR dye-labeled lectin for the detection of early neoplasia in BE in an ex vivo setting.Methods Consecutive patients undergoing endoscopic mucosal resection (EMR) for BE-related early neoplasia were recruited. Freshly collected EMR specimens were sprayed at the bedside with fluorescent lectin and then imaged. Punch biopsies were collected from each EMR under NIR light guidance. We compared the fluorescence intensity from dysplastic and nondysplastic areas within EMRs and from punch biopsies with different histological grades.Results 29 EMR specimens were included from 17 patients. A significantly lower fluorescence was found for dysplastic regions across whole EMR specimens (P < 0.001). We found a 41 % reduction in the fluorescence of dysplastic compared to nondysplastic punch biopsies (P < 0.001), with a sensitivity and specificity for dysplasia detection of 80 % and 82.9 %, respectively.Conclusion Lectin-based NIR imaging can differentiate dysplastic from nondysplastic Barrett’s mucosa ex vivo. The incidence of esophageal adenocarcinoma (EAC) has increased dramatically in the Western world over the last 30 years. Despite treatment advances this cancer is still associated with a 5-year survival of less than 15 %, due principally to late diagnosisEndoscopic surveillance is generally performed according to the Seattle protocol, which entails 4 biopsies every 2 centimeters within the BE segment, but is affected by sampling error due to inconspicuous dysplasia and sampling errorThe combination of molecular imaging tools with endoscopy may provide novel routes for better diagnosisHere we describe the synthesis and characterization of a novel, topically applied lectin-based probe for the endoscopic detection of early BE-related neoplasia, based on the conjugation of a lectin, wheat germ agglutinin (WGA), to a commercially available and clinically applicable NIR fluorophore (IR800CW)The study was approved by the Cambridgeshire-2 Research Ethics Committee (09/H0308/118).Consecutive patients, referred for endoscopic management of Barrett’s-related neoplasia, were recruited prospectively between June 2014 and February 2016 at a single tertiary referral center  . ExclusGastroscopies were carried out using FQ260Z endoscopes . The neoplastic areas were assessed by a combination of white light endoscopy, narrow band imaging with magnification, and autofluorescence imaging for precise delineation of the lesion. EMR was carried out with a Duette multiband mucosectomy device .WGA , was conjugated to IR800CW-NHS ester dye , and purified using modified versions of the methods described elsewhereEMR specimens were collected from patients, washed with 5 mL of ice-cold phosphate-buffered saline (PBS), sprayed with 2 mL of WGA-IR800 (10 μg/mL) in PBS, incubated in the dark for 10 min at room temperature, and then washed again with the same buffer, prior to imaging using an intraoperative fluorescence imaging device . A maximum of two punch biopsies (2 mm diameter) were collected ex vivo from each EMR specimen under NIR fluorescence guidance. The combined staining, imaging, biopsy collection procedure was limited to a maximum time of 30 minutes, to ensure preservation of the specimens for subsequent histopathology. Following the procedure, EMR specimens and biopsies were immediately fixed in formalin.The EMR specimen paraffin block was cut at 2-mm intervals from the 12-o’clock to the 6-o’clock margin and single sections were mounted onto glass slides. Single sections were read every 1 mm, in a direction orthogonal to the original cut, by the pathologist.Cases were reviewed by two pathologists with experience in upper GI neoplasia, including the study pathologist (M.O’D.) who has extensive experience in reporting Barrett’s esophageal neoplasia and a satisfactory level of interobserver agreement with external pathologists in previous studiesExcluded from the analysis were EMR specimens with no dysplasia or less than 3 dysplastic/neoplastic grid elements and EMR specimens that failed orientation.Ftest. GraphPad Prism  was used throughout in the analysis.P < 0.05 was considered statistically significant.Data are expressed as mean ± SD, unless stated otherwise. The two-tailed Wilcoxon matched-pairs signed rank test was used for pairwise comparison of dysplastic and nondysplastic areas of EMR specimens. The Mann–Whitney test was used for analysis of punch biopsy fluorescence. Correlation analysis was performed using the extra sum-of-squaresBecause of the exploratory nature of this ex vivo imaging study and the absence of a previously acquired large dataset, a formal calculation of sample size was not possible. We anticipated that with an average of 50 data points per EMR specimen from the pathology grids , 20 EMRA total of 21 patients were recruited into the study. After exclusion of 4 patients who presented with advanced lesions not amenable to EMR, 17 patients completed the endoscopy and ex vivo imaging protocol. The median age was 65 years (range 52 – 84), the average length of the Barrett’s segment was 4.2 cm (range 0.5 – 10 cm), and the median lesion size was 1 cm (range 1 – 4 cm). On average, 2 EMR specimens (range 1 – 5) were collected per patient, from which a median of 2 punch biopsies were collected per EMR (range 0 – 2).The fluorescence signal from EMR specimens, collected at the bedside and freshly stained with WGA-IR800, was analyzed in relation to pathology. Co-registration with the pathology grid allowed correlation of the histopathology for each specimen level and for each grid cell with the corresponding NIR fluorescence signal. We included 29 EMR specimens in this analysis  .P < 0.001) in comparison with areas that showed no dysplasia ( The mean fluorescence intensity (MFI) values of areas at similar pathological stages were calculated for the multiple EMR specimens collected from individual patients /ND × 100, whereDandNDare the average MFIs for grid elements containing dysplastic and nondysplastic regions, respectively. This ratio reflects the contrast in fluorescence signal between dysplastic and nondysplastic areas within an EMR specimen, and corrects for differences in absolute fluorescence signal intensities that arose from inconsistent spraying of the EMR specimens on different days. By definition it could only be calculated for EMRs containing dysplasia.Negative contrast (%) between areas of dysplasia and nondysplasia was defined as the ratio  was found between the negative contrast associated with dysplastic vs. nondysplastic regions within an EMR specimen and the corresponding spatial extent of dysplasia. Representative specimens are shown, containing low  than biopsies collected from regions with nondysplastic Barrett’s epithelium. A receiver operating characteristic (ROC) analysis (P < 0.001), with a sensitivity of 80 % and a specificity of 82.9 %.IMC can normally be detected based on standard high definition white light endoscopy. For this reason, and because the main aim of the technique is to improve detection of early dysplastic lesions (namely LGD or HGD), we excluded biopsies containing neoplasia (IMC). In a pooled analysis , biopsinalysis , with aP < 0.001) than biopsies collected from regions with nondysplastic BE. ROC analysis indicated an AUC, sensitivity, and specificity of 0.79 ± 0.06 (P < 0.001), 75.0 % and 70.7 % respectively.Biopsies collected from regions that did contain cancer, also had a significantly lower MFI  between nondysplastic and dysplastic tissue was also confirmed for a large cohort of punch biopsies  criteria for detection of early neoplasia in Barrett’s esophagus


Poecilia reticulata) habituate to repeated exposure to a mild stressor, using a waterborne hormone sampling approach that has previously been shown to elicit a stress response in small fish. We also test for individual variation in the extent of habituation to this stressor. Concentrating on freely circulating cortisol, we found that the first exposure to the assay induced high cortisol release rates but that guppies tended to habituate quickly to subsequent exposures. There were consistent differences among individuals in their average cortisol release rate (after accounting for effects of variables such as body size) over repeated exposures. Our analyses did not find evidence of individual differences in habituation rate, although limitations in statistical power could account for this finding. We repeated the analysis for free 11-ketotestosterone, which can also respond to stressors, but found no obvious habituation pattern and no among-individual variation. We also present data on conjugated forms of both hormones, which were repeatable but did not show the expected time-lagged habituation effect. We discuss consistent individual differences around the general pattern of habituation in the flexible stress response, and highlight the potential for individual variation in habituation to facilitate selection against the deleterious effects of chronic stress.The vertebrate stress response enables individuals to react to and cope with environmental challenges. A crucial aspect of the stress response is the elevation of circulating glucocorticoids. However, continued activation of the stress response under repeated exposure to stressors can be damaging to fitness. Under certain circumstances it may therefore be adaptive to habituate to repeated exposures to a particular stressor by reducing the magnitude of any associated release of glucocorticoids. Here, we investigate whether Trinidadian guppies ( While dAmong-individual variation has now been documented in behavioural  and neurHabituation can be defined as occurring where repeated, or prolonged, application of a stimulus results in a progressively weaker stress response . IntuitiPoecilia reticulata. We focus principally on HPI activity and thus cortisol, the major GC in teleost fish  Act 1986 under licence from the Home Office (UK), and with local ethical approval from the University of Exeter.The guppies used in this study were taken from our captive population, descended from wild fish collected in 2008 from the lower Aripo River, Trinidad, and housed at the University of Exeter’s Penryn campus. We sampled 32 adult fish  from a stock tank haphazardly, and tagged each for identification purposes with coloured elastomer injection under mild MS222 sedation. We housed fish in 2 mixed-sex groups of equal size, using separate tanks that shared a common recirculating water supply. All fish were fed to satiation twice daily (0800–1000 h and again at 1600–1800 h) using commercial flake food and live 2.2We used a waterborne sampling method  to obtaiImmediately after sampling, we filtered the water samples  and then passed them through solid phase C18 extraction columns  that had been primed with 2 × 2 ml HPLC-grade methanol followed by 2 × 2 ml distilled water, keeping the column moist prior to sample extraction. We used high-purity tubing  to pass the water samples to the columns, through which they were drawn slowly under vacuum pressure (see We later shipped the frozen columns on dry ice from the University of Exeter’s Penryn campus to the University of Alabama. We thawed each column and purged with 2 × 2 ml distilled water before eluting both free and conjugated (sulphated and glucuronidated) hormones. We used 2 × 2 ml ethyl acetate and 2 × 2 ml HPLC-grade methanol in successive, separate washes to elute free and conjugated hormones respectively  into sepBriefly, 1 ml trifluoroacetic acid [TFA]:ethyl acetate (1:100) was added to the hormone residue and incubated overnight in a water bath at 37 °C. The TFA:ethyl acetate was evaporated under nitrogen at 37 °C followed by addition of 0.5 ml 0.1 M sodium acetate buffer (pH 4.5). We vortexed the sample for 5 min, followed by addition of 4 ml diethyl ether and a further 5 min of vortexing. We then allowed samples to sit for approximately 30 min to enable separation of the organic and aqueous phases, and then immersed the samples in a methanol-dry ice bath to freeze the aqueous phase. The organic phase containing the sulphated fraction was poured off into a new 13 × 100 mm borosilicate vial. 10 µl of β-glucuronidase  was added to the remaining aqueous phase and incubated overnight in a water bath at 37 °C, followed by the diethyl ether extraction as described above. The organic phase containing the glucuronidated fraction was poured into the same vial as the ether containing the sulphated fraction to produce a sample with total conjugated hormone. Diethyl ether was evaporated under nitrogen in a 37 °C water bath and the hormone residue was resupended in 200 µl of 5% EtOH: 95% EIA kit buffer followed by vortexing for 20 min. We ran assays in strict accordance with the manufacturer’s instructions, and all samples were run in duplicate.12 = 0.204, P = 0.84; Cortisol-2: t12 = 0.013, P = 0.99; 11KT-1: t12 = 0.02, P = 0.98; 11KT-2: t12 = 0.005, P = 0.99). We also ran the pooled samples in duplicate at both the beginning and end of each of the four 96-well plates for each hormone (free fraction) to assess intra- and inter-assay coefficients of variation. Intra-assay coefficients of variation were 6.9%, 11.5%, 4.9%, and 9.5% for each of the cortisol plates and 7.8%, 3.4%, 2.9%, and 1.6% for each of the 11KT plates; the inter-assay coefficients for cortisol and 11KT were 10.7% and 7.3%, respectively. We generated a separate pool for the conjugated hormone, which was run in duplicate at the beginning and end of an additional four 96-well plates for each hormone (conjugated fraction). Intra-assay coefficients of variation were 4.9%, 7.7%, 6.6% and 5.6% for cortisol and 5.6%, 15.2%, 7.6% and 10.1% for 11KT; the inter-assay coefficients for cortisol and 11KT were 6.5% and 11.3%, respectively.We generated a pooled waterborne hormone extract for males and females separately by combining 20 µl taken from each of the 128 samples. These pools were serially diluted to assess parallelism with the standard curves from the Cayman Chemicals, Inc cortisol and 11KT kits. Two serial dilution curves were run in duplicate for each hormone, and each was parallel to the standard curve .In what follows we conceptualise habituation as plasticity in endocrine state with respect to repeated stressor exposure. This allows us to test for and characterise among-individual variation in habituation using the well-known ‘character state’ and ‘reaction norm’ frameworks for analysing plasticity . In the The analysis procedure described below was performed separately for both free and conjugated forms of cortisol (log nanograms) and 11-ketotestosterone (log picograms). We present only the results for free hormones below, with results and discussion of conjugated forms available in the 2.3.1free, 11KTfree, Cortisolconjugate, 11KTconjugate) in turn, we tested for the hypothesised effects of habituation on hormone levels across repeated assays (model A) by fitting assay repeat . We also included sex, and the interaction of sex and assay repeat to account for any sex difference in mean endocrine state or in average habituation. We specified assay repeat as a factor rather than as a continuous predictor to allow maximum flexibility . Tank , sampling order , and body mass were included as further covariates to control for potential sources of variance not relevant to our present hypotheses. We standardised body mass measurements by centring and scaling (subtracting the population mean and dividing by the standard deviation) to improve their interpretability . For each response trait we use the simplified fixed effects structure as determined by the model used to test for habituation described above. To test for repeatable differences in average hormone levels  across all four repeats, we compared a ‘null’ model  to one in which we fit a random effect of individual ID (B2). We then extended the random intercepts model to include among-individual differences in habituation rate , by fitting both a random ID effect and a random interaction of ID with assay repeat number (this time as a continuous covariate), as well as the intercept-slope covariance (model B3). Model B3 is identical to the simplified form of model A. We compared nested models  using likelihood ratio tests (LRTs), in which we assume that twice the difference in model log-likelihoods conforms to a chi-square distribution where the degrees of freedom are set by the number of additional parameters in the more complex model. When testing the effect of a single variance term, the test statistic distribution is assumed to correspond to a 50:50 mix of chi-squared distributions having 0 and 1 degrees of freedom respectively .r1,2 > r1,3 > r1,4)).Second, for each response variable we formulated a multivariate (4-‘trait’) model to test hypotheses about variance in – and covariance among – the four repeat-specific observations (models C1-C3). Rather than using the raw data, we estimated (co)variances conditional on the repeat-specific means as well as on the fixed effects as described above . We fitt33.13, 63.3 = 0.63, P = 0.60). In contrast, male and female guppies do differ in how their 11KT levels change across repeated assays  across the four successive stress exposures. Free cortisol concentration follows a pattern that largely conforms to our expectations of habituation, the mean declining significantly from stress exposures 1 to 2 and remaining stable at 3, although then showing a slight increase at exposure 4 A. Female3.220,1 = 3.11, P = 0.04). Individuals do not show significant differences in their average level of free 11KT .LRT comparison of the univariate mixed models with and without a random effect of individual identity provides statistical support for repeatable differences in average free cortisol concentrations across the repeated stress exposures. That said, the adjusted repeatability  for free cortisol is relatively low . Under this random slope model, the correlation between individual intercepts and slopes is strongly positive , although the standard errors around the estimate are large (r = 0.88 SE 0.56). For free 11KT we were unable to detect any estimable variance in random slope .Addition of random reaction norm slope terms to the mixed models does not lead to a significantly better model for either free cortisol or free 11KT. For free cortisol, visual inspection of model predictions is certainly suggestive of an improved fit to the observed data B,C, thou3.323 = 8.2, P = 0.03; 26 = 14.0, P = 0.03), indicative of some repeatable variation among individuals. However, examination of the covariance-correlation matrix , and no evidence for significant within-individual covariance structure  exposure relative to later ones. Our analyses of among-individual variation – conducted within both random regression and multivariate “character state” frameworks – also provide clear evidence that individuals differ in their average stress-induced hormone levels across sampling events. While we find some limited support for our final prediction that individuals would also differ in their rates of habituation with regard to stress-induced free cortisol release, statistical evidence is equivocal .4.1The high levels of (average) free cortisol during the first stress exposure, coupled with the decreased levels during subsequent exposures, are consistent with our prediction that habituation should occur with repeated stressor exposure. The population-level habituation dynamics seen here echo largely those reported previously in guppies  and otheDanio rerio), although in these cases with higher cortisol found in males relative to females , as a linear effect of body mass was not significant in our model of free cortisol . The sex difference in stress responsive cortisol levels in guppies could therefore reflect different selection pressures acting on the male and female stress response, possibly due to sex differences in life histories, rather than simple associations with body size . For exa females . Rambo eSalmo trutta, Male guppies in our study have higher free 11KT concentrations than females across all stress exposures, in line with the androgenic role of this hormone . However4.2In addition to the population-level evidence of habituation reported above, we also find evidence of consistent differences among individuals in their average stress-induced free cortisol concentrations . Whether there are differences among individuals in their rate of habituation to the stressor is less clear. Plotting fitted reaction norms shows that individuals seem to diverge over repeated stress exposures, yet allowing for the existence of individual differences in these habituation gradients did not yield a model of significantly better fit to the data than assuming that all individuals shared the same habituation gradient. Similarly, the character state approach shows that individual variance increases over successive repeats; however, the covariance structure is not consistently positive, suggesting that this is not a simple case of individuals habituating at different rates. That these patterns are suggestive of individual variation in habituation, but without yielding conclusive evidence for it, points to a lack of sufficient power in our study. Mixed effects models – particularly those with complex random effects structures – can be data hungry, and it appears likely that a greater number of individuals would need to be assayed in order to demonstrate that there are significant differences among individuals in their rate of habituation to a repeated stressor . In contOne of the main drivers behind the interest in among-individual variation in labile traits is that its presence means there is an opportunity for selection to act on them. Such variation can stem from a variety of different sources, which have been discussed widely in the behavioural literature e.g.,  and are 4.3As predicted, we find that guppies habituate quickly to repeated exposure to a stressor. We also provide examples of a framework for characterising GC variability and flexibility within and among individuals using both reaction norm and character state approaches. Our results show individual variation in stress-induced cortisol levels, in addition to some limited evidence of individual variation in habituation to the stressor. Such variation provides material for selection to act upon, and could (given underlying genetic variation in habituation rate) enable a population to mount an evolutionary response – for example, to the deleterious effects of a failure to habituate to repeated or prolonged exposure to a stressor. Given the current lack of consensus around how to diagnose chronic stress in populations , one fru


P) or recovery efficiency (%R) and the concentrations of ligands and nickel species were analysed using Winhumic V software. A negative correlation between P and the concentration of organic nickel complexes was found, suggesting that only labile nickel species are transported through the liquid membrane, with non-labile complexes remaining in the water sample; allowing for their separation and subsequent quantification in natural waters.The different species of nickel present in natural waters exhibit different transport behaviour through bulk liquid membranes (BLMs). This fact has been used to design and optimise a separation/pre-concentration system applicable to separate labile and non-labile nickel fractions. A hydrazone derivative—1,2-cyclohexanedione bis-benzoyl-hydrazone  dissolved in toluene/dimethyl formamide (2% DMF)—was used as a chemical carrier of nickel species, from an aqueous source solution (sample) to a receiving acidic solution. Both chemical and hydrodynamic conditions controlling the transport system were studied and optimised. Under optimum conditions, variations in the transport of nickel ions as a function of organic (humic acids) and inorganic (chloride ions) ligands were studied. Relationships between the permeability coefficient ( By using AAS, the potential interferences caused by other transported metals can be overcame.−1) and NaCl (5–25 g·L−1 Cl−) were added to different source solutions containing 1 mg·L−1 nickel, in order to evaluate the effects of both organic and inorganic ligands on the transport of nickel through the membrane. Both, the permeability coefficient and recovery efficiency (measured as the percentage of nickel recovered in the receiving solution) were estimated as a function of ligand concentrations. To confirm the results obtained with organic matter, experiments with humic acids were repeated but, before membrane experiments, organic matter was removed by oxidation with UV radiation for 90 min using an A705 UV digester , after the addition of 100 µL H2O2 to each sample. Permeability coefficients were calculated after UV digestion and compared with values obtained in the presence of organic matter.HA (15–100 mg·L−1 Ni2+) real seawater sample from the Gulf of Cádiz after a UV digestion was performed to evaluate the influence of sample matrix on the recovery efficiency.Analysis of a spiked .−1) is shown in P value was reached for 2.5 mmol·L−1 1,2-CHBBH and then lower P value was observed, together with microprecipiation in the organic phase. Therefore, this concentration of 1,2-CHBBH was selected for further experiments.The dependence of the permeability coefficient of nickel on 1,2-CHBBH concentration  10−3 cm·min−1 was obtained. If chloride ions were present in the samples, a decrease in P was observed due to the formation of monovalent NiCl+ complexes, which could exhibit a lower transport rate or even not be extracted by 1,2-CHBBH. When Cl− concentrations varied between 5 and 25 g·L−1, P value was nearly independent of Cl− concentration and a mean permeability coefficient of 5.64 (±0.36) 10−3 cm·min−1 was obtained. For seawater conditions, the observed decrease in permeability coefficient was 26%, consistent with the concentration of NiCl+ in the samples  10−3 cm·min−1 for 15–100 mg·L−1 HA.As shown in − and HA mostly resulted in the formation of NiCl+ and anionic Ni-HA complexes which could be not transported through the liquid membrane. Permeability coefficients significantly correlated (α = 0.05) with the concentration of organic complexes, with a Spearman coefficient of 0.943.In addition to P, the recovery efficiency of transport was also calculated for different concentrations of humic acids. Although the time required to achieve maximum transport was not affected by the presence of humic acids, a decrease in the efficiency was observed, as can be seen in As a consequence of these results, we concluded that only the most labile nickel species are transported through the membrane, while stable complexes, mainly organic complexes, remain in the water sample, enabling separation of the different fractions.A significant negative correlation (α = 0.05) was observed between the recovery efficiency and the concentration of organic matter, measured both as dissolved organic carbon (DOC) and Ni-HA complexes, with a Spearman coefficient of −0.900. Based on these results, a linear relationship was established to estimate the recovery efficiency (%R) as a function of DOC:Finally, the difference between labile and total nickel concentrations can be used to estimate the non-labile nickel fraction.−1 1,2-CHBBH in the organic phase; 1.5 mol·L−1 HCl in the receiving solution; and a stirring rate of 500 rpms. Under these conditions, the permeability coefficient and recovery efficiency were dependent on the humic acid concentration, resulting in a decrease in nickel transport as ligand concentration increased. This decrease was related to the formation of non-labile complexes between nickel and humic acids, which cannot be transported through the membrane, as evidenced by the correlation between the permeability coefficient and theoretical NiHA concentrations obtained from Winhumic V software. Our results suggest that the liquid membrane system can be applied to separate labile and non-labile nickel fractions in natural water samples, even seawater. Speciation could be performed after only one extraction of the nickel present in the samples and previous analyses of the dissolved organic carbon concentration.We evaluated a novel bulk liquid membrane system to separate nickel fractions from natural waters. Optimum conditions to maximise nickel transport through the membrane were determined to be: 2.5 mmol·L


After 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS) was injected, additional capacitance changes due to the smaller molecular weight Aβ oligomers disassembled from the higher molecular weight oligomeric Aβ were determined (ΔCoAβ). By dividing two values, the capacitance ratio (ΔCoAβ/ΔCtAβ) was determined and then normalized to the capacitance change index (CCI). The CCI was higher in the ADD group than in the OND (p = 0.040) and CU groups (p = 0.007). The accuracy of the CCI was fair in separating into the ADD and CU groups . These results demonstrate that the level of Aβ in nasal secretions increases in ADD and the detection of Aβ in nasal secretions using IME biosensors may be possible in predicting ADD.We investigated the level of amyloid beta (Aβ) in nasal secretions of patients with Alzheimer’s disease dementia (ADD) using interdigitated microelectrode (IME) biosensors and determined the predictive value of Aβ in nasal secretions for ADD diagnosis. Nasal secretions were obtained from 35 patients with ADD, 18 with cognitive decline associated with other neurological disorders (OND), and 26 cognitively unimpaired (CU) participants. Capacitance changes in IMEs were measured by capturing total Aβ (ΔC Current therapies such as acetylcholinesterase inhibitors and the N-methyl-D-aspartate receptor antagonist memantine may provide relief with respect to symptoms, but they do not treat the underlying process of AD. Neuropathological hallmarks of AD include amyloid β (Aβ)-containing plaques and tau-containing neurofibrillary tangles, which are found throughout the brain2. These hallmarks have become the main target of current drug development. However, several drug candidates that target Aβ have not been successful in large, multicentre clinical trials5. It is now generally acknowledged that this is partly because a large proportion of clinically diagnosed patients show no evidence of amyloid pathology on positron emission tomography (PET) scans5. Recent advances in AD research also show that amyloid pathology begins 20–30 years before the clinical onset of AD6. These findings indicate the need for validated biomarkers in drug development and clinical practice.Alzheimer’s disease (AD) is the most common cause of dementia, as it accounts for 60 to 70% of the overall prevalence of dementia. AD is a neurodegenerative disorder characterized by an insidious onset and a progressive deterioration in cognition, functional ability, and behaviour9 and low cerebrospinal fluid (CSF) Aβ4211. Biomarkers of fibrillar tau include elevated CSF phosphorylated tau (p-tau) and cortical tau PET ligands13. Biomarkers of neurodegeneration or neuronal injury are CSF total tau (t-tau)13, 18fluorodeoxyglucose PET hypometabolism, and atrophy on magnetic resonance imaging (MRI)15. However, PET and MRI are expensive, and CSF biomarker analysis requires a lumbar puncture, which is an invasive procedure with potential adverse side effects such as headache and back pain. Interlaboratory variability of CSF biomarker analysis is also high. Recently, the results of studies of blood-based biomarkers of AD pathology such as plasma Aβ and tau have been reported19. Simple and inexpensive tests for blood-based biomarkers would be desirable due to their safety and minimal invasiveness. However, given the lack of consistency of most blood-based biomarkers across studies, validation in another cohort is needed, and other cost-effective and feasible methods for the early diagnosis of AD are needed.In the last decade, remarkable progress has been achieved in the study of AD biomarkers. Biomarkers of Aβ plaques include cortical amyloid PET ligands20. It has been reported that in AβPP/PS1 transgenic mice, the deposition of Aβ began in the olfactory system and then spread to the hippocampus and cortex21. In experiments in rats, ventricle-injected isotope-labelled Aβ peptide (125I-Aβ40) was shown to be transported to the nasal cavity through a non-haematogenous pathway22. In another recent study, median levels of p-tau/t-tau ratios in the middle nasal meatus and in the olfactory cleft were significantly higher in AD cases than in controls, but the levels of Aβ42 and Aβ40 were near zero and were not different between AD cases and controls23. However, the methods used in the study may not have detected a very low level of Aβ in nasal secretions.In a recent study, older adults with olfactory dysfunction were more than twice as likely to develop dementia 5 years later, and more odour identification errors were associated with a greater probability of an interval dementia diagnosisIn this study, we investigated the level of Aβ in nasal secretions of patients with AD dementia (ADD) using interdigitated microelectrode (IME) methods and examined the predictive value of Aβ in nasal secretions for the diagnosis of ADD.24 and CDR-Sum of Boxes (SB) scores were significantly different among the groups after adjusted for age. The MMSE (p < 0.001), and CDR-SB (p < 0.001) scores were significantly lower in the ADD group than in the CU group. The MMSE (p = 0.003) and CDR-SB (p < 0.001) scores were also significantly lower in the ADD group than in the OND group. Finally, the MMSE (p < 0.001) and CDR-SB (p < 0.001) scores were significantly lower in the OND group than in the CU group.Nasal secretions were obtained from 35 patients with ADD, 18 patients with cognitive decline associated with other neurological disorders (OND), and 26 cognitively unimpaired (CU) participants. Table p = 0.040) and CU groups (p = 0.007)  in nasal secretions was significantly different among the groups after adjusted for age Table . Bonferr07) Fig. . The CCI07) Fig. .Table 2Cp = 0.004) for the diagnosis of ADD among the participants of the ADD and CU groups  for the diagnosis of ADD among the participants of the ADD and OND groups  curve was used to examine the predictive value of the CCI in nasal secretions for a diagnosis of ADD. The area under the ROC curve (AUC) was 0.718 -1-piperazinepropanesulfonic acid (EPPS)-treated samples according to time  were analyzed by western blots. Figure 25. More studies are needed before Aβ in nasal section can be used as a biomarker for AD. The following studies may be needed to improve accuracy. First, the amount of nasal secretions collected varied among the participants, so the concentration of Aβ needs to be considered. Second, if there is a diurnal variation in the amount of Aβ in nasal secretions, it is necessary to collect them from participants at similar times. Third, we need to consider using a more powerful substance than EPPS to disaggregate high molecular weight Aβ. Fourth, more advanced measurement system may be needed.In this study, the CCI in nasal secretions was elevated in the patients with ADD compared to the CU individuals and those with OND. The CCI in nasal secretions is greater when the level of soluble Aβ in nasal secretions is higher. Therefore, the present results demonstrate that the soluble Aβ such as oligomeric Aβ in nasal secretions is elevated in ADD patients compared to CU individuals and those with OND. The present results demonstrate the possibility that the ultra-sensitive detection of soluble Aβ in nasal secretions using IME biosensors may be effective in predicting ADD. However, the accuracy of the CCI as measured by the AUC was fair in separating into the ADD and CU groups. The accuracy of CCI in nasal secretions for the prediction of ADD was lower than that of amyloid PET ligands and CSF AD biomarkers found in previous studies26. In the future, the CCI should be evaluated only in OND patients with amyloid-negative results on PET or CSF studies.The accuracy of the CCI in nasal secretions was also nearly fair to distinguish between ADD and OND patients. To be a diagnostic marker for ADD, it is important that a biomarker is able to distinguish ADD from patients with other diseases as well as from normal individuals. Although the results of this study are still lacking, the present results suggest that if accuracy improves, the CCI reflecting the amount of soluble Aβ in nasal secretions may be useful in distinguishing ADD from other causes of dementia. Although the presence or absence of amyloid deposition using PET or CSF studies was found in about 82% of the participants in this study, some participants of the OND group may also have had multiple brain pathologies including AD27. This result suggests that Aβ in body fluids may measure different aspects of AD Aβ pathology from those of amyloid PET.The CCI was significantly correlated with global cognitive function scales. Higher CCI was related to lower global cognitive function, and thus the CCI may reflect the natural course of AD. However, whether a change in the CCI is associated with cognitive decline should be evaluated in a longitudinal observational study. No correlation was observed between CSF and PET amyloid value changes in a longitudinal study28. Previously, we had failed to measure Aβ in nasal secretions by Luminex multiplex assay. Electrical impedimetric biosensors have attracted attention due to their label-free and real-time monitoring of the morphological and physiological changes in DNA, protein, and cells29. For the electrical impedance measurement of the specimens, micro- or nano-sized electrode-based sensors are used. The IME biosensor could therefore be a method by which ultra-low levels of proteins are measured in body fluids.The ultra-sensitive detection of small amounts of Aβ in nasal secretions could be performed using IME methods31. In a previous report, olfactory impairment in cognitively normal elderly was associated with incident amnestic mild cognitive impairment (aMCI) and progression from aMCI to ADD over a mean 3.5 years of follow-up32. This study suggests that olfactory deterioration may precede cognitive decline in AD. More research is needed on the olfactory function and measurement of Aβ in nasal secretions in AD.The previous clinical studies and a meta-analysis showed that patients with ADD had the evidence for olfactory deficits33 were disassembled to several numbers of lower molecular weight Aβ oligomers . Thus, it looks clear that the increase of target molecules is closely related to the increase of capacitance change (ΔCoAβ) and furthermore, clinical effectiveness of CCI for ADD diagnosis based on the nasal secretions. The reason why other bands of disassembled Aβ oligomers except a band at ~70 kDa were not clearly shown in the blot image might have been that their concentrations were too low to be detectable by western blots. The western blot analysis did not provide any information on the isoforms of Aβ oligomers (40 and 42 or their mixture) captured by 6E10 anti-Aβ antibody as well. Nevertheless, Aβ42 can be the most reasonable candidate among the amyloid proteins which are contained in discrete bands. In a previous study, authors have used a western blot analysis, to which 6E10 antibody applied, for the characterization of high molecular weight Aβ42 oligomers as well as its monomer isolated from human hippocampal samples34.The study of EPPS-derived disassembly of soluble Aβ by the western blots demonstrated that soluble Aβ proteins such as high molecular weight Aβ oligomer42 levels. Some patients without brain amyloid pathology may have been included in the ADD group, while other individuals with brain amyloid pathology may have been included in the CU and OND groups. Third, we did not examine the associations of the CCI in nasal secretions with other AD biomarkers such as amyloid PET, CSF Aβ42, t-tau and p-tau levels, and cortical thickness. Fourth, we did not directly measure the levels of Aβ in nasal secretions, but rather, we measured the Aβ levels in nasal secretions indirectly by assessing changes in capacitance. Fifth, we did not pinpoint the species of Aβ detected by 6E10 anti-Aβ antibody in the baseline nasal secretions as well as in the nasal secretions after disaggregation by EPPS. In the future, it is necessary to define the size and isoforms of Aβ using mass spectrometry. It is also necessary to analyze nasal secretion with other anti-Aβ antibodies, such as antibodies targeting oligomeric Aβ42. Sixth, we did not investigate the efficiency of examining the amount of EPPS that allows the CCI to reach its maximum value35.Our study had several limitations. First, the results should be interpreted with caution due to the small sample size. The results of this study should be reproduced in other studies with large sample sizes. Second, amyloid biomarkers such as amyloid PET ligands and CSF biomarkers were not studied for all participants, although approximately 82% of participants had historical results available for amyloid PET or CSF AβTo our knowledge, this is the first study to show that Aβ levels in nasal secretions are elevated in ADD patients compared to CU individuals and those with OND. The results of this study are still preliminary but suggest that CCI reflecting the amount of soluble Aβ in nasal secretions, such as oligomeric Aβ, may be useful in predicting ADD if accuracy improves. More studies are needed before Aβ in nasal secretions can be used as a biomarker for AD.36. The OND group consisted of nine patients with vascular cognitive impairment37, three patients with frontotemporal lobar degeneration38, one patient with Parkinson’s disease39, one patient with epilepsy, and four patients with other dementia.From October 2015 to August 2018, 38 patients with ADD, 18 patients with cognitive decline associated with OND, and 27 CU elderly controls participated in this study. Three patients with ADD and one CU individual withdrew their consent. Finally, nasal secretions were obtained from 79 participants, who ranged in age from 50 to 90 years. We recruited patients with ADD or OND from the memory clinic at Inha University Hospital, Incheon, South Korea. The participants with ADD met the criteria for probable ADD according to the National Institute on Aging-Alzheimer’s Association core clinical criteria40: stroke or transient ischaemic attack, seizures, Parkinson’s disease, multiple sclerosis, cerebral palsy, Huntington’s disease, encephalitis, meningitis, brain surgery, surgery to clear arteries to the brain, diabetes that requires insulin to control, hypertension that is not well controlled, cancer other than skin cancer diagnosed within the past three years, shortness of breath while sitting still, use of oxygen at home, heart attack that resulted in changes in memory, walking or problem solving lasting at least 24 hours, kidney dialysis, liver disease, hospitalization for mental or emotional problems within the past five years, current use of medications for mental or emotional problems, alcohol consumption greater than three drinks per day, drug abuse in the past five years, treatment for alcohol abuse in the past five years, unconsciousness for more than one hour other than during surgery, overnight hospitalization because of a head injury, illness causing a permanent decrease in memory or other mental functions, trouble with vision that prevents reading ordinary print even with glasses, and difficulty understanding conversations because of hearing even if wearing a hearing aid. They also had MMSE scores more than 1.5 standard deviation below the age- and education adjusted normative means24.The CU individuals were recruited from patients who visited the neurology clinic at the same hospital for other reasons . They did not have any of the 28 diseases associated with decreases in cognitive function or a history suggestive of a decrease in cognitive functionWe excluded participants who took medications that affect nasal function within 1 week of the study entry, those who underwent nasal surgery within 3 months, those who were diagnosed with rhinitis or sinusitis by simple X-ray or computed tomography scan, those with a history of exposure to chemical stimulants, current smokers, those with severe or uncontrolled medical disease, and those who were pregnant or lactating.41. They were also administered a brain MRI and routine biochemical and serological tests that included a thyroid function test, tests for serum vitamin B12 and folate levels, and a Venereal Disease Research Laboratory test.All participants underwent comprehensive physical and neurological examinations, including an extensive standardized neuropsychological battery, the Seoul Neuropsychological Screening Battery (SNSB)18F-flutemetamol PET imaging, nine with positive 18F-florbetaben PET imaging, three with positive ¹¹C-Pittsburgh compound-B (11C-PIB) PET imaging, and one with positive 18F-florbetapir PET imaging  participants and by CSF Aβ measurements in two participants. Evidence of brain amyloid pathology in the ADD group was demonstrated in fifteen patients with positive The study was performed in accordance with the International Harmonization Conference guidelines on Good Clinical Practice and was approved by the institutional review board of Inha University Hospital prior to study initiation. Prior to participation in the study, all participants provided written informed consent for their participation in the study. The legal representatives of the participants with dementia also provided written informed consent.et al.42 A Merocel® sponge was placed into both nasal cavities of the patient for 1 minute. At this stage, the sponge was placed towards the nasal roof so that it was as close to the olfactory bulb as possible. After the sponge was removed from the participant’s nose, we inserted it in a specially shaped tube with a funnel. When the sponge is placed on the funnel in this tube, the nasal secretion absorbed by the sponge flows down through the funnel which is designed to be tucked down. We performed centrifugation at 4 °C and 5000 rpm for 10 minutes to isolate all nasal secretions from the sponge. The nasal secretions were then stored at a temperature of −80 °C until further analysis.The collection of nasal secretions was performed according to the method previously reported by Cho c-ToAD, Cantis Inc., South Korea) used to analyse human nasal secretions is briefly composed of (i) c-ToAD detection system, which is a customized impedance analyser with 8 device docking sockets and a syringe pump, (ii) the workstation computer for data acquisition from the c-ToAD detection system, the process of which is related to visualization and motion control of the syringe pump connected to the c-ToAD cartridge, and (iii) the disposable c-ToAD cartridge. All communications between the c-ToAD detection system including the micro-pump and the computer were achieved via Bluetooth wireless technology  and PBS (×3) sequentially by repeating the flow of the wash buffers. Capacitance changes in the IMEs were measured by capturing the molecules of total Aβ at this step . After the measurement, EPPS was carefully injected into the microfluidic channels, and simultaneously, the second capacitance measurement was performed . The capacitance change over time at the post-EPPS step indicates that monomeric or lower molecular weight Aβ molecules that disassembled from oligomeric Aβ by EPPS were gradually associated with the unoccupied antigen binding fragment (Fab) binding sites of the Aβ antibody   to adjust their viscosity, after which they were contained in a 200 μL syringe. By driving the microsyringe pump, a maximum of 16 samples was carefully delivered to the microfluidic channels of the c-ToAD detection system were spontaneously transmitted to the c-ToAD workstation via wireless transmission during measurement. In principle, the c-ToAD software averaged all of capacitances obtained from the 8-IME array in a single microfluidic channel according to an electric parameter setting and algorithm and calculated capacitance changes of the total Aβ (ΔCtAβ) at the pre-EPPS step and those of the oligomeric Aβ (ΔCoAβ) at the post-EPPS step to yield the capacitance ratio (ΔCoAβ/ΔCtAβ) of each sample. Finally, all of ΔCoAβ/ΔCtAβ values were normalized to specific indices between 0 and 99 according to a measurement standard ).As mentioned above, all data measured by the http://rsbweb.nih.gov/ij/index.html).Nasal secretions collected from 7 ADD patients with amyloid deposition on PET and 8 CU individuals without amyloid deposition on PET were used in the western blot analysis. Nasal secretions were lysed in ice-cold RIPA buffer containing 1× proteinase inhibitor cocktail. Lysate was incubated in ice for 20 minutes and centrifuged at 14,000 r.p.m., 4 °C for 30 min. The supernatant was used in Aβ western blots for biochemical changes. Protein samples (40 μg) were loaded on each lane of SDS–PAGE gels to detect Aβ oligomer, disassembled lower molecular Aβ oligomer and β-actin (a loading control). Afterward, the blots were probed with anti-Aβ (6E10) or anti-β-actin antibodies (1:1000) at 4 °C overnight, followed by incubation with secondary goat anti-mouse horse radish peroxidase-conjugated immunoglobulin G  at room temperature for 1 h. The signals were visualized using enhanced chemiluminescence. The band intensity was quantified on ImageJ software (Characteristic data were presented as means and standard deviations (SD) for continuous variables and frequency and percentages for categorical variables. The analysis of variance (ANOVA) was used to compare age and education among the groups. When a statistical significant overall difference was detected in the ANOVA test, pairwise comparisons on means between diagnosis groups were conducted with Tukey method. For the categorical variables, namely, gender, CDR, APOE ε4 carrier status and Aβ deposition on PET, we calculated the frequencies and compared their differences across the groups using the χ² test. The analysis of covariance (ANCOVA) with age as a covariate was used to compare MMSE, CDR-SB, and CCI among the groups. When a statistical significant overall difference was detected in the ANCOVA test, pairwise comparisons on means between diagnosis groups were conducted with Bonferroni post hoc analysis. The ANOVA and Bonferroni post hoc analysis were used to compare the amounts of proteins according to the time in the western blot. Spearman’s rho was used to measure the correlations between CCI and global cognitive function. The sensitivity and specificity of the CCI for the diagnosis of ADD were evaluated by ROC curve analysis. The cutoff values were determined to yield the best Youden index (sensitivity + specificity − 1) for ADD diagnosis. Significance for all tests was set at α = 0.05, two-tailed. All statistical analyses were performed using SPSS 19.0 .Supplementary Information