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John G Love,1 John S McKenzie,2 Efsevia A Nikokavoura,3 John Broom,3 Catherine Rolland,3 Kelly L Johnston4,5 1School of Applied Social Studies, Faculty of Health & Social Care, Robert Gordon University, Aberdeen, UK; 2Rowett Institute of Health & Nutrition, University of Aberdeen, St Mary’s, Kings College, Aberdeen, UK; 3Centre for Obesity Research, Faculty of Health & Social Care, Robert Gordon University, Aberdeen, UK; 4LighterLife, Harlow, Essex, UK; 5Diabetes and Nutritional Sciences Division, Faculty of Health and Medical Sciences, Kings College London, London, UK Abstract: Polycystic ovary syndrome (PCOS) is variously reported to affect between 5% and 26% of reproductive age women in the UK and accounts for up to 75% of women attending fertility clinics due to anovulation. The first-line treatment option for overweight/obese women with PCOS is diet and lifestyle interventions. However, optimal dietary guidelines are missing, with very little research having been done in this area. This paper presents the findings from a qualitative study (using semistructured interviews) of ten obese women who had PCOS and who had used LighterLife Total (LLT), a commercial weight loss program which utilizes a very low-calorie diet in conjunction with behavioral change therapy underpinned by group support. We investigated the women’s history of obesity, their experiences of other diets compared with LLT, and the on-going impact that this has had on their lives. Findings show that most women reported greater success using this weight loss program in terms of achieving and maintaining weight loss when compared with other diets. Furthermore, all the women nominated LLT as their model weight loss intervention with only a few modifications. Keywords: PCOS, obesity, weight loss, diet

Fast and reliable identification of bacteria directly in clinical samples is a critical factor in clinical microbiological diagnostics. Current approaches require time-consuming bacterial isolation and enrichment procedures, delaying stratified treatment. Here, we describe a biomarker-based strategy that utilises bacterial small molecular metabolites and lipids for direct detection of bacteria in complex samples using mass spectrometry (MS). A spectral metabolic library of 233 bacterial species is mined for markers showing specificity at different phylogenetic levels. Using a univariate statistical analysis method, we determine 359 so-called taxon-specific markers (TSMs). We apply these TSMs to the in situ detection of bacteria using healthy and cancerous gastrointestinal tissues as well as faecal samples. To demonstrate the MS method-agnostic nature, samples are analysed using spatial metabolomics and traditional bulk-based metabolomics approaches. In this work, TSMs are found in >90% of samples, suggesting the general applicability of this workflow to detect bacterial presence with standard MS-based analytical methods., Competing Interests: Competing interests: The authors declare no competing interest related to this work., (© 2024. The Author(s).)

Breast cancer, a complex disease with a significant prevalence to form metastases, necessitates novel therapeutic strategies to improve treatment outcomes. Here, we present the results of a comparative molecular study of primary breast tumours, their metastases, and the corresponding primary cell lines using Desorption Electrospray Ionisation (DESI) and Laser-Assisted Rapid Evaporative Ionisation Mass Spectrometry (LA-REIMS) imaging. Our results show that ambient ionisation mass spectrometry technology is suitable for rapid characterisation of samples, providing a lipid- and metabolite-rich spectrum within seconds. Our study demonstrates that the lipidomic fingerprint of the primary tumour is not significantly distinguishable from that of its metastasis, in parallel with the similarity observed between their respective primary cell lines. While significant differences were observed between tumours and the corresponding cell lines, distinct lipidomic signatures and several phospholipids such as PA(36:2), PE(36:1), and PE(P-38:4)/PE(O-38:5) for LA-REIMS imaging and PE(P-38:4)/PE(O-38:5), PS(36:1), and PI(38:4) for DESI-MSI were identified in both tumours and cells. We show that the tumours' characteristics can be found in the corresponding primary cell lines, offering a promising avenue for assessing tumour responsiveness to therapeutic interventions. A comparative analysis by DESI-MSI and LA-REIMS imaging revealed complementary information, demonstrating the utility of LA-REIMS in the molecular imaging of cancer.

Basal cell carcinoma (BCC), the most common keratinocyte cancer, presents a substantial public health challenge due to its high prevalence. Traditional diagnostic methods, which rely on visual examination and histopathological analysis, do not include metabolomic data. This exploratory study aims to molecularly characterize BCC and diagnose tumour tissue by applying matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) and machine learning (ML). BCC tumour development was induced in a mouse model and tissue sections containing BCC (n = 12) were analysed. The study design involved three phases: (i) Model training, (ii) Model validation and (iii) Metabolomic analysis. The ML algorithm was trained on MS data extracted and labelled in accordance with histopathology. An overall classification accuracy of 99.0% was reached for the labelled data. Classification of unlabelled tissue areas aligned with the evaluation of a certified Mohs surgeon for 99.9% of the total tissue area, underscoring the model's high sensitivity and specificity in identifying BCC. Tentative metabolite identifications were assigned to 189 signals of importance for the recognition of BCC, each indicating a potential tumour marker of diagnostic value. These findings demonstrate the potential for MALDI-MSI coupled with ML to characterize the metabolomic profile of BCC and to diagnose tumour tissue with high sensitivity and specificity. Further studies are needed to explore the potential of implementing integrated MS and automated analyses in the clinical setting., (© 2024 The Author(s). Experimental Dermatology published by John Wiley & Sons Ltd.)

Cutaneous squamous cell carcinoma (SCC) is an increasingly prevalent global health concern. Current diagnostic and surgical methods are reliable, but they require considerable resources and do not provide metabolomic insight. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) enables detailed, spatially resolved metabolomic analysis of tissue samples. Integrated with machine learning, MALDI-MSI could yield detailed information pertaining to the metabolic alterations characteristic for SCC. These insights have the potential to enhance SCC diagnosis and therapy, improving patient outcomes while tackling the growing disease burden. This study employs MALDI-MSI data, labelled according to histology, to train a supervised machine learning model (logistic regression) for the recognition and delineation of SCC. The model, based on data acquired from discrete tumor sections (n = 25) from a mouse model of SCC, achieved a predictive accuracy of 92.3% during cross-validation on the labelled data. A pathologist unacquainted with the dataset and tasked with evaluating the predictive power of the model in the unlabelled regions, agreed with the model prediction for over 99% of the tissue areas. These findings highlight the potential value of integrating MALDI-MSI with machine learning to characterize and delineate SCC, suggesting a promising direction for the advancement of mass spectrometry techniques in the clinical diagnosis of SCC and related keratinocyte carcinomas., (© 2024. The Author(s).)

Extracellular vesicles (EVs), including exosomes and microvesicles, mediate intercellular communication in cancer, from development to metastasis. EV-based liquid biopsy is a promising strategy for cancer diagnosis as EVs can be found in cancer patients' body fluids. In this study, the lipid composition of breast cancer-derived EVs was studied as well as the potential of blood plasma EVs for the identification of lipid biomarkers for breast cancer detection. Initially, an untargeted lipidomic analysis was carried out for a panel of cancerous and non-cancerous mammary epithelial cells and their secreted EVs. We found that breast cancer-derived EVs are enriched in sphingolipids and glycerophospholipids compared to their parental cells. The initial in vitro study showed that EVs and their parental cells can be correctly classified (100% accuracy) between cancerous and non-cancerous, as well as into their respective breast cancer subtypes, based on their lipid composition. Subsequently, an untargeted lipidomic analysis was carried out for blood plasma EVs from women diagnosed with breast cancer (primary or progressive metastatic breast cancer) as well as healthy women. Correspondingly, when blood plasma EVs were analysed, breast cancer patients and healthy women were correctly classified with an overall accuracy of 93.1%, based on the EVs' lipid composition. Similarly, the analysis of patients with primary breast cancer and healthy women showed an overall accuracy of 95% for their correct classification. Furthermore, primary and metastatic breast cancers were correctly classified with an overall accuracy of 89.5%. This reveals that the blood plasma EVs' lipids may be a promising source of biomarkers for detection of breast cancer. Additionally, this study demonstrates the usefulness of untargeted lipidomics in the study of EV lipid composition and EV-associated biomarker discovery studies. This is a proof-of-concept study and a starting point for further analysis on the identification of EV-based biomarkers for breast cancer., (© 2024 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

Objective: Rapid evaporative ionization mass spectrometry (REIMS) is a metabolomic technique analyzing tissue metabolites, which can be applied intraoperatively in real-time. The objective of this study was to profile the lipid composition of colorectal tissues using REIMS, assessing its accuracy for real-time tissue recognition and risk-stratification., Summary Background Data: Metabolic dysregulation is a hallmark feature of carcinogenesis; however, it remains unknown if this can be leveraged for real-time clinical applications in colorectal disease., Methods: Patients undergoing colorectal resection were included, with carcinoma, adenoma and paired-normal mucosa sampled. Ex vivo analysis with REIMS was conducted using monopolar diathermy, with the aerosol aspirated into a Xevo G2S QToF mass spectrometer. Negatively charged ions over 600 to 1000 m/z were used for univariate and multivariate functions including linear discriminant analysis., Results: A total of 161 patients were included, generating 1013 spectra. Unique lipidomic profiles exist for each tissue type, with REIMS differentiating samples of carcinoma, adenoma, and normal mucosa with 93.1% accuracy and 96.1% negative predictive value for carcinoma. Neoplasia (carcinoma or adenoma) could be predicted with 96.0% accuracy and 91.8% negative predictive value. Adenomas can be risk-stratified by grade of dysplasia with 93.5% accuracy, but not histological subtype. The structure of 61 lipid metabolites was identified, revealing that during colorectal carcinogenesis there is progressive increase in relative abundance of phosphatidylglycerols, sphingomyelins, and mono-unsaturated fatty acid-containing phospholipids., Conclusions: The colorectal lipidome can be sampled by REIMS and leveraged for accurate real-time tissue recognition, in addition to riskstratification of colorectal adenomas. Unique lipidomic features associated with carcinogenesis are described., Competing Interests: Conflicts of interest: Z.T. acts as a consultant for waters and is the inventor of REIMS. No other potential conflicts of interest exist., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

In this study, we examine the suitability of desorption electro-flow focusing ionization (DEFFI) for mass spectrometry imaging (MSI) of biological tissue. We also compare the performance of desorption electrospray ionization (DESI) with and without the flow focusing setup. The main potential advantages of applying the flow focusing mechanism in DESI is its rotationally symmetric electrospray jet, higher intensity, more controllable parameters, and better portability due to the robustness of the sprayer. The parameters for DEFFI have therefore been thoroughly optimized, primarily for spatial resolution but also for intensity. Once the parameters have been optimized, DEFFI produces similar images to the existing DESI. MS images for mouse brain samples, acquired at a nominal pixel size of 50 μm, are comparable for both DESI setups, albeit the new sprayer design yields better sensitivity. Furthermore, the two methods are compared with regard to spectral intensity as well as the area of the desorbed crater on rhodamine-coated slides. Overall, the implementation of a flow focusing mechanism in DESI is shown to be highly suitable for imaging biological tissue and has potential to overcome some of the shortcomings experienced with the current geometrical design of DESI.

Optical microscopy has long been the gold standard to analyse tissue samples for the diagnostics of various diseases, such as cancer. The current diagnostic workflow is time-consuming and labour-intensive, and manual annotation by a qualified pathologist is needed. With the ever-increasing number of tissue blocks and the complexity of molecular diagnostics, new approaches have been developed as complimentary or alternative solutions for the current workflow, such as digital pathology and mass spectrometry imaging (MSI). This study compares the performance of a digital pathology workflow using deep learning for tissue recognition and an MSI approach utilising shallow learning to annotate formalin-fixed and paraffin-embedded (FFPE) breast cancer tissue microarrays (TMAs). Results show that both deep learning algorithms based on conventional optical images and MSI-based shallow learning can provide automated diagnostics with F1-scores higher than 90%, with the latter intrinsically built on biochemical information that can be used for further analysis.

The ability to adapt to low-nutrient microenvironments is essential for tumor cell survival and progression in solid cancers, such as colorectal carcinoma (CRC). Signaling by the NF-κB transcription factor pathway associates with advanced disease stages and shorter survival in patients with CRC. NF-κB has been shown to drive tumor-promoting inflammation, cancer cell survival, and intestinal epithelial cell (IEC) dedifferentiation in mouse models of CRC. However, whether NF-κB affects the metabolic adaptations that fuel aggressive disease in patients with CRC is unknown. Here, we identified carboxylesterase 1 (CES1) as an essential NF-κB-regulated lipase linking obesity-associated inflammation with fat metabolism and adaptation to energy stress in aggressive CRC. CES1 promoted CRC cell survival via cell-autonomous mechanisms that fuel fatty acid oxidation (FAO) and prevent the toxic build-up of triacylglycerols. We found that elevated CES1 expression correlated with worse outcomes in overweight patients with CRC. Accordingly, NF-κB drove CES1 expression in CRC consensus molecular subtype 4 (CMS4), which is associated with obesity, stemness, and inflammation. CES1 was also upregulated by gene amplifications of its transcriptional regulator HNF4A in CMS2 tumors, reinforcing its clinical relevance as a driver of CRC. This subtype-based distribution and unfavorable prognostic correlation distinguished CES1 from other intracellular triacylglycerol lipases and suggest CES1 could provide a route to treat aggressive CRC.

Background: There is a paucity of population-based data on chlamydia in pregnancy despite rising rates in US women. Our objectives were to assess chlamydia prevalence by age group and to identify factors associated with infection in pregnant women to inform screening guidelines., Methods: This cross-sectional study included pregnant women tested for chlamydia who delivered at the University of Alabama at Birmingham between November 1, 2012, and December 31, 2017. The primary outcome was chlamydia prevalence, defined as a positive urogenital chlamydia nucleic acid amplification test result documented in the electronic medical record. Multivariable logistic regression was used to identify factors associated with infection., Results: Among 17,796 women who delivered during the study period, 13,657 (77%) had chlamydia testing performed at the University of Alabama at Birmingham. Chlamydia prevalence (95% confidence interval) was 7.4% (7.0%-7.9%). Age-stratified prevalence rates were 14.6%, 4.3%, and 1.7% for women younger than 25 years, 25 to 29 years, and 30 years or older, respectively. Chlamydia in pregnancy remained strongly associated with age (adjusted odds ratio [95% confidence interval], 7.2 [5.6-9.2] for age <25 years, and 2.3 [1.7-3.0] for ages 25-29 years, when compared with >30 years) after adjustment for race, urban residence, and insurance status., Conclusions: Among pregnant women living in the southeastern United States, chlamydia was detected in 1 of 14 women who were tested. Chlamydia positivity was highest among women younger than 30 years. Study findings support broad screening for chlamydia in pregnancy.

The incidence of esophageal adenocarcinoma is rising, survival remains poor, and new tools to improve early diagnosis and precise treatment are needed. Cancer phospholipidomes quantified with mass spectrometry imaging (MSI) can support objective diagnosis in minutes using a routine frozen tissue section. However, whether MSI can objectively identify primary esophageal adenocarcinoma is currently unknown and represents a significant challenge, as this microenvironment is complex with phenotypically similar tissue-types. Here, we used desorption electrospray ionization-MSI (DESI-MSI) and bespoke chemometrics to assess the phospholipidomes of esophageal adenocarcinoma and relevant control tissues. Multivariate models derived from phospholipid profiles of 117 patients were highly discriminant for esophageal adenocarcinoma both in discovery (AUC = 0.97) and validation cohorts (AUC = 1). Among many other changes, esophageal adenocarcinoma samples were markedly enriched for polyunsaturated phosphatidylglycerols with longer acyl chains, with stepwise enrichment in premalignant tissues. Expression of fatty acid and glycerophospholipid synthesis genes was significantly upregulated, and characteristics of fatty acid acyls matched glycerophospholipid acyls. Mechanistically, silencing the carbon switch ACLY in esophageal adenocarcinoma cells shortened glycerophospholipid chains, linking de novo lipogenesis to the phospholipidome. Thus, DESI-MSI can objectively identify invasive esophageal adenocarcinoma from a number of premalignant tissues and unveils mechanisms of phospholipidomic reprogramming. SIGNIFICANCE: These results call for accelerated diagnosis studies using DESI-MSI in the upper gastrointestinal endoscopy suite, as well as functional studies to determine how polyunsaturated phosphatidylglycerols contribute to esophageal carcinogenesis., (©2020 American Association for Cancer Research.)

Background: Rabies is endemic in southern Bhutan, associated with 1-2 human deaths and high post exposure prophylaxis (PEP) costs annually. Evaluation of clinicians' management of human cases potentially exposed to rabies could contribute to improving PEP prescribing practices to both reduce unnecessary costs associated with PEP and reach the target of zero human deaths due to rabies by 2023., Methods: A cross-sectional survey of 50 clinicians' management of human cases potentially exposed to rabies was conducted in 13 health centers in high-rabies-risk areas of Bhutan during February-March 2016., Results: Data were collected on clinicians' management of 273 human cases potentially exposed to rabies. The 50 clinicians comprised health assistants or clinical officers (55%) and medical doctors (45%) with a respective median of 19, 21 and 2 years' experience. There was poor agreement between clinicians' rabies risk assessment compared with an independent assessment for each case based on criteria in the National Rabies Management Guidelines (NRMG). Of the 194 cases for which clinicians recorded a rabies risk category, only 53% were correctly classified when compared with the NRMG. Clinicians were more likely to underestimate the risk of exposure to rabies and appeared to prescribe PEP independently of their risk classification.. Male health assistants performed the most accurate risk assessments while female health assistants performed the least accurate. Clinicians in Basic Health Units performed less accurate risk assessments compared with those in hospitals., Conclusions: This study highlights important discrepancies between clinicians' management of human cases potentially exposed to rabies and recommendations in the NRMG. In particular, clinicians were not accurately assessing rabies risk in potentially exposed cases and were not basing their PEP treatment on the basis of their risk assessment. This has significant implications for achieving the national goal of eliminating dog-mediated human rabies by 2030 and may result in unnecessary costs associated with PEP. Recommendations to improve clinician's management of human cases potentially exposed to rabies include: reviewing and updating the NRMG, providing clinicians with regular and appropriately targeted training about rabies risk assessment and PEP prescription, and regularly reviewing clinicians' practices.

Leptospirosis is an important zoonotic disease in Nepal; however, there is a lack of information on sources of leptospirosis infection for people and associated risk factors. We implemented a case-control study nested within a cross-sectional survey to investigate zoonotic risks of human leptospirosis among adult, febrile patients seeking treatment in healthcare centres in Kaski District, Nepal, from April to October 2013. The study population was 239 febrile patients; the data consisted of paired blood samples; questionnaires on clinical symptoms; occupational, environmental and animal exposures; and a blood sample from animals in the household. In total, 63 cattle, 92 buffalo, 181 goats, 20 dogs and 14 rodents from 119 households were blood sampled. Serology was performed on the samples using the microscopic agglutination test (MAT) for a panel of 20 serovars with a starting dilution of 1:50. Clinical leptospirosis was defined as a titre of 1:400 or stronger, or a 4-fold or greater rise between acute and convalescent titres, or evidence of seroconversion for one or more serovars represented by a titre of <1:50 in the acute sample and a titre of ≥1:100 in the convalescent sample. The prevalence of clinical leptospirosis was 5.4% (95% CI 2.6%-8.3%). The most common symptoms among recruited participants were loss of appetite and lethargy followed by chills, profuse sweating and backache. No individual clinical symptom nor combination of any two symptoms was significantly associated with clinical leptospirosis. However, owning goats (OR 1.3, CI 95% 1.05-1.66), working in rice fields (OR 1.3, CI 95% 1.11-1.72) and male gender (OR 4, CI 95% 1.12-17.26) significantly increased the risk of clinical leptospirosis in humans. Findings suggest that leptospirosis should be considered in the clinical differential diagnosis among febrile adult patients in Nepal, especially for men, goat owners and/or those who work in rice fields., (© 2018 Blackwell Verlag GmbH.)

Rodents are well-known reservoirs and vectors of many emerging and re-emerging infectious diseases, but little is known about their role in zoonotic disease transmission in Bhutan. In this study, a cross-sectional investigation of zoonotic disease pathogens in rodents was performed in Chukha district, Bhutan, where a high incidence of scrub typhus and cases of acute undifferentiated febrile illness had been reported in people during the preceding 4-6 months. Twelve rodents were trapped alive using wire-mesh traps. Following euthanasia, liver and kidney tissues were removed and tested using PCR for Orientia tsutsugamushi and other bacterial and rickettsial pathogens causing bartonellosis, borreliosis, human monocytic ehrlichiosis, human granulocytic anaplasmosis, leptospirosis, and rickettsiosis. A phylogenetic analysis was performed on all rodent species captured and pathogens detected. Four out of the 12 rodents (33.3%) tested positive by PCR for zoonotic pathogens. Anaplasma phagocytophilum, Bartonella grahamii, and B. queenslandensis were identified for the first time in Bhutan. Leptospira interrogans was also detected for the first time from rodents in Bhutan. The findings demonstrate the presence of these zoonotic pathogens in rodents in Bhutan, which may pose a risk of disease transmission to humans.

Background: Survival from ovarian cancer (OC) is improved with surgery, but surgery can be complex and tumour identification, especially for borderline ovarian tumours (BOT), is challenging. The Rapid Evaporative Ionisation Mass Spectrometric (REIMS) technique reports tissue histology in real-time by analysing aerosolised tissue during electrosurgical dissection., Methods: Aerosol produced during diathermy of tissues was sampled with the REIMS interface. Histological diagnosis and mass spectra featuring complex lipid species populated a reference database on which principal component, linear discriminant and leave-one-patient-out cross-validation analyses were performed., Results: A total of 198 patients provided 335 tissue samples, yielding 3384 spectra. Cross-validated OC classification vs separate normal tissues was high (97·4% sensitivity, 100% specificity). BOT were readily distinguishable from OC (sensitivity 90.5%, specificity 89.7%). Validation with fresh tissue lead to excellent OC detection (100% accuracy). Histological agreement between iKnife and histopathologist was very good (kappa 0.84, P < 0.001, z = 3.3). Five predominantly phosphatidic acid (PA(36:2)) and phosphatidyl-ethanolamine (PE(34:2)) lipid species were identified as being significantly more abundant in OC compared to normal tissue or BOT (P < 0.001, q < 0.001)., Conclusions: The REIMS iKnife distinguishes gynaecological tissues by analysing mass-spectrometry-derived lipidomes from tissue diathermy aerosols. Rapid intra-operative gynaecological tissue diagnosis may improve surgical care when histology is unknown, leading to personalised operations tailored to the individual.

Analyzing lipid composition and distribution within the brain is important to study white matter pathologies that present focal demyelination lesions, such as multiple sclerosis. Some lesions can endogenously re-form myelin sheaths. Therapies aim to enhance this repair process in order to reduce neurodegeneration and disability progression in patients. In this context, a lipidomic analysis providing both precise molecular classification and well-defined localization is crucial to detect changes in myelin lipid content. Here we develop a correlated heterospectral lipidomic (HSL) approach based on coregistered Raman spectroscopy, desorption electrospray ionization mass spectrometry (DESI-MS), and immunofluorescence imaging. We employ HSL to study the structural and compositional lipid profile of demyelination and remyelination in an induced focal demyelination mouse model and in multiple sclerosis lesions from patients ex vivo . Pixelwise coregistration of Raman spectroscopy and DESI-MS imaging generated a heterospectral map used to interrelate biomolecular structure and composition of myelin. Multivariate regression analysis enabled Raman-based assessment of highly specific lipid subtypes in complex tissue for the first time. This method revealed the temporal dynamics of remyelination and provided the first indication that newly formed myelin has a different lipid composition compared to normal myelin. HSL enables detailed molecular myelin characterization that can substantially improve upon the current understanding of remyelination in multiple sclerosis and provides a strategy to assess remyelination treatments in animal models., Competing Interests: The authors declare no competing financial interest.

A new, more robust sprayer for desorption electrospray ionization (DESI) mass spectrometry imaging is presented. The main source of variability in DESI is thought to be the uncontrolled variability of various geometric parameters of the sprayer, primarily the position of the solvent capillary, or more specifically, its positioning within the gas capillary or nozzle. If the solvent capillary is off-center, the sprayer becomes asymmetrical, making the geometry difficult to control and compromising reproducibility. If the stiffness, tip quality, and positioning of the capillary are improved, sprayer reproducibility can be improved by an order of magnitude. The quality of the improved sprayer and its potential for high spatial resolution imaging are demonstrated on human colorectal tissue samples by acquisition of images at pixel sizes of 100, 50, and 20 μm, which corresponds to a lateral resolution of 40-60 μm, similar to the best values published in the literature. The high sensitivity of the sprayer also allows combination with a fast scanning quadrupole time-of-flight mass spectrometer. This provides up to 30 times faster DESI acquisition, reducing the overall acquisition time for a 10 mm × 10 mm rat brain sample to approximately 1 h. Although some spectral information is lost with increasing analysis speed, the resulting data can still be used to classify tissue types on the basis of a previously constructed model. This is particularly interesting for clinical applications, where fast, reliable diagnosis is required. Graphical Abstract ᅟ.

Background: Re-operation for positive resection margins following breast-conserving surgery occurs frequently (average = 20-25%), is cost-inefficient, and leads to physical and psychological morbidity. Current margin assessment techniques are slow and labour intensive. Rapid evaporative ionisation mass spectrometry (REIMS) rapidly identifies dissected tissues by determination of tissue structural lipid profiles through on-line chemical analysis of electrosurgical aerosol toward real-time margin assessment., Methods: Electrosurgical aerosol produced from ex-vivo and in-vivo breast samples was aspirated into a mass spectrometer (MS) using a monopolar hand-piece. Tissue identification results obtained by multivariate statistical analysis of MS data were validated by histopathology. Ex-vivo classification models were constructed from a mass spectral database of normal and tumour breast samples. Univariate and tandem MS analysis of significant peaks was conducted to identify biochemical differences between normal and cancerous tissues. An ex-vivo classification model was used in combination with bespoke recognition software, as an intelligent knife (iKnife), to predict the diagnosis for an ex-vivo validation set. Intraoperative REIMS data were acquired during breast surgery and time-synchronized to operative videos., Results: A classification model using histologically validated spectral data acquired from 932 sampling points in normal tissue and 226 in tumour tissue provided 93.4% sensitivity and 94.9% specificity. Tandem MS identified 63 phospholipids and 6 triglyceride species responsible for 24 spectral differences between tissue types. iKnife recognition accuracy with 260 newly acquired fresh and frozen breast tissue specimens (normal n = 161, tumour n = 99) provided sensitivity of 90.9% and specificity of 98.8%. The ex-vivo and intra-operative method produced visually comparable high intensity spectra. iKnife interpretation of intra-operative electrosurgical vapours, including data acquisition and analysis was possible within a mean of 1.80 seconds (SD ±0.40)., Conclusions: The REIMS method has been optimised for real-time iKnife analysis of heterogeneous breast tissues based on subtle changes in lipid metabolism, and the results suggest spectral analysis is both accurate and rapid. Proof-of-concept data demonstrate the iKnife method is capable of online intraoperative data collection and analysis. Further validation studies are required to determine the accuracy of intra-operative REIMS for oncological margin assessment.

Visual inspection of tumour tissues does not reveal the complex metabolic changes that differentiate cancer and its sub-types from healthy tissues. Mass spectrometry imaging, which quantifies the underlying chemistry, represents a powerful tool for the molecular exploration of tumour tissues. A 3-dimensional topological description of the chemical properties of the tumour permits the formulation of hypotheses about the biological composition and interactions and the possible causes of its heterogeneous structure. The large amount of information contained in such datasets requires powerful tools for its analysis, visualisation and interpretation. Linear methods for unsupervised dimensionality reduction, such as PCA, are inadequate to capture the complex non-linear relationships present in these data. For this reason, a deep unsupervised neural network based technique, parametric t-SNE, is adopted to map a 3D-DESI-MS dataset from a human colorectal adenocarcinoma biopsy onto a 2-dimensional manifold. This technique allows the identification of clusters not visible with linear methods. The unsupervised clustering of the tumour tissue results in the identification of sub-regions characterised by the abundance of identified metabolites, making possible the formulation of hypotheses to account for their significance and the underlying biological heterogeneity in the tumour.

Ambient ionization mass spectrometry was developed as a sample preparation-free alternative to traditional MS-based workflows. Desorption electrospray ionization (DESI)-MS methods were demonstrated to allow the direct analysis of a broad range of samples including unaltered biological tissue specimens. In contrast to this advantageous feature, nowadays DESI-MS is almost exclusively used for sample preparation intensive mass spectrometric imaging (MSI) in the area of cancer research. As an alternative to MALDI, DESI-MSI offers matrix deposition-free experiment with improved signal in the lower (<500m/z) range. DESI-MSI enables the spatial mapping of tumor metabolism and has been broadly demonstrated to offer an alternative to frozen section histology for intraoperative tissue identification and surgical margin assessment. Rapid evaporative ionization mass spectrometry (REIMS) was developed exclusively for the latter purpose by the direct combination of electrosurgical devices and mass spectrometry. In case of the REIMS technology, aerosol particles produced by electrosurgical dissection are subjected to MS analysis, providing spectral information on the structural lipid composition of tissues. REIMS technology was demonstrated to give real-time information on the histological nature of tissues being dissected, deeming it an ideal tool for intraoperative tissue identification including surgical margin control. More recently, the method has also been used for the rapid lipidomic phenotyping of cancer cell lines as it was demonstrated in case of the NCI-60 cell line collection., (© 2017 Elsevier Inc. All rights reserved.)

Ovarian cancer is highly prevalent among European women, and is the leading cause of gynaecological cancer death. Current histopathological diagnoses of tumour severity are based on interpretation of, for example, immunohistochemical staining. Desorption electrospray mass spectrometry imaging (DESI-MSI) generates spatially resolved metabolic profiles of tissues and supports an objective investigation of tumour biology. In this study, various ovarian tissue types were analysed by DESI-MSI and co-registered with their corresponding haematoxylin and eosin (H&E) stained images. The mass spectral data reveal tissue type-dependent lipid profiles which are consistent across the n = 110 samples (n = 107 patients) used in this study. Multivariate statistical methods were used to classify samples and identify molecular features discriminating between tissue types. Three main groups of samples (epithelial ovarian carcinoma, borderline ovarian tumours, normal ovarian stroma) were compared as were the carcinoma histotypes (serous, endometrioid, clear cell). Classification rates >84% were achieved for all analyses, and variables differing statistically between groups were determined and putatively identified. The changes noted in various lipid types help to provide a context in terms of tumour biochemistry. The classification of unseen samples demonstrates the capability of DESI-MSI to characterise ovarian samples and to overcome existing limitations in classical histopathology.

Introduction: Considerable advocacy, funding, training, and technical support have been provided to South Asian countries to strengthen One Health (OH) collaborative approaches for controlling diseases with global human pandemic potential since the early 2000s. It is essential that the OH approach continues to be strengthened given South Asia is a hot spot for emerging and endemic zoonotic diseases. The objectives of this article are to describe OH research and training and capacity building activities and the important developments in government support for OH in these countries to identify current achievements and gaps., Materials and Methods: A landscape analysis of OH research, training, and government support in South Asia was generated by searching peer-reviewed and grey literature for OH research publications and reports, a questionnaire survey of people potentially engaged in OH research in South Asia and the authors' professional networks., Results: Only a small proportion of zoonotic disease research conducted in South Asia can be described as truly OH, with a significant lack of OH policy-relevant research. A small number of multisectoral OH research and OH capacity building programmes were conducted in the region. The governments of Bangladesh and Bhutan have established operational OH strategies, with variable progress institutionalising OH in other countries. Identified gaps were a lack of useful scientific information and of a collaborative culture for formulating and implementing integrated zoonotic disease control policies and the need for ongoing support for transdisciplinary OH research and policy-relevant capacity building programmes., Discussion: Overall we found a very small number of truly OH research and capacity building programmes in South Asia. Even though significant progress has been made in institutionalising OH in some South Asian countries, further behavioural, attitudinal, and institutional changes are required to strengthen OH research and training and implementation of sustainably effective integrated zoonotic disease control policies., Competing Interests: and funding The authors have not received any funding or benefits from industry or elsewhere to conduct this study.

Histopathological assessment of lymph node metastases (LNM) depends on subjective analysis of cellular morphology with inter-/intraobserver variability. In this study, LNM from esophageal adenocarcinoma was objectively detected using desorption electrospray ionization-mass spectrometry imaging (DESI-MSI). Ninety lymph nodes (LN) and their primary tumor biopsies from 11 esophago-gastrectomy specimens were examined and analyzed by DESI-MSI. Images from mass spectrometry and corresponding histology were coregistered and analyzed using multivariate statistical tools. The MSIs revealed consistent lipidomic profiles of individual tissue types found within LNs. Spatial mapping of the profiles showed identical distribution patterns as per the tissue types in matched IHC images. Lipidomic profile comparisons of LNM versus the primary tumor revealed a close association in contrast to benign LN tissue types. This similarity was used for the objective prediction of LNM in mass spectrometry images utilizing the average lipidomic profile of esophageal adenocarcinoma. The multivariate statistical algorithm developed for LNM identification demonstrated a sensitivity, specificity, positive predictive value, and negative predictive value of 89.5%, 100%, 100%, and 97.2%, respectively, when compared with gold-standard IHC. DESI-MSI has the potential to be a diagnostic tool for perioperative identification of LNM and compares favorably with techniques currently used by histopathology experts. Cancer Res; 76(19); 5647-56. ©2016 AACR., (©2016 American Association for Cancer Research.)

Rapid evaporative ionization mass spectrometry (REIMS) was used for the rapid mass spectrometric profiling of cancer cell lines. Spectral reproducibility was assessed for three different cell lines, and the extent of interclass differences and intraclass variance was found to allow the identification of these cell lines based on the REIMS data. Subsequently, the NCI60 cell line panel was subjected to REIMS analysis, and the resulting data set was investigated for its distinction of individual cell lines and different tissue types of origin. Information content of REIMS spectral profiles of cell lines were found to be similar to those obtained from mammalian tissues although pronounced differences in relative lipid intensity were observed. Ultimately, REIMS was shown to detect changes in lipid content of cell lines due to mycoplasma infection. The data show that REIMS is an attractive means to study cell lines involving minimal sample preparation and analysis times in the range of seconds.

In this study, the impact of sprayer design and geometry on performance in desorption electrospray ionization mass spectrometry (DESI-MS) is assessed, as the sprayer is thought to be a major source of variability. Absolute intensity repeatability, spectral composition, and classification accuracy for biological tissues are considered. Marked differences in tissue analysis performance are seen between the commercially available and a lab-built sprayer. These are thought to be associated with the geometry of the solvent capillary and the resulting shape of the primary electrospray. Experiments with a sprayer with a fixed solvent capillary position show that capillary orientation has a crucial impact on tissue complex lipid signal and can lead to an almost complete loss of signal. Absolute intensity repeatability is compared for five lab-built sprayers using pork liver sections. Repeatability ranges from 1 to 224% for individual sprayers and peaks of different spectral abundance. Between sprayers, repeatability is 16%, 9%, 23%, and 34% for high, medium, low, and very low abundance peaks, respectively. To assess the impact of sprayer variability on tissue classification using multivariate statistical tools, nine human colorectal adenocarcinoma sections are analyzed with three lab-built sprayers, and classification accuracy for adenocarcinoma versus the surrounding stroma is assessed. It ranges from 80.7 to 94.5% between the three sprayers and is 86.5% overall. The presented results confirm that the sprayer setup needs to be closely controlled to obtain reliable data, and a new sprayer setup with a fixed solvent capillary geometry should be developed.

Background: Three-dimensional (3D) imaging mass spectrometry (MS) is an analytical chemistry technique for the 3D molecular analysis of a tissue specimen, entire organ, or microbial colonies on an agar plate. 3D-imaging MS has unique advantages over existing 3D imaging techniques, offers novel perspectives for understanding the spatial organization of biological processes, and has growing potential to be introduced into routine use in both biology and medicine. Owing to the sheer quantity of data generated, the visualization, analysis, and interpretation of 3D imaging MS data remain a significant challenge. Bioinformatics research in this field is hampered by the lack of publicly available benchmark datasets needed to evaluate and compare algorithms., Findings: High-quality 3D imaging MS datasets from different biological systems at several labs were acquired, supplied with overview images and scripts demonstrating how to read them, and deposited into MetaboLights, an open repository for metabolomics data. 3D imaging MS data were collected from five samples using two types of 3D imaging MS. 3D matrix-assisted laser desorption/ionization imaging (MALDI) MS data were collected from murine pancreas, murine kidney, human oral squamous cell carcinoma, and interacting microbial colonies cultured in Petri dishes. 3D desorption electrospray ionization (DESI) imaging MS data were collected from a human colorectal adenocarcinoma., Conclusions: With the aim to stimulate computational research in the field of computational 3D imaging MS, selected high-quality 3D imaging MS datasets are provided that could be used by algorithm developers as benchmark datasets.

In Bhutan, Capture-Neuter-Vaccinate-Release (CNVR) programs have been implemented to manage the dog population and control rabies, but no detailed evaluation has been done to assess their coverage and impact. We compared estimates of the dog population using three analytical methods: Lincoln-Petersen index, the Chapman estimate, and the logit-normal mixed effects model, and a varying number of count periods at different times of the day to recommend a protocol for applying the mark-resight framework to estimate free-roaming dog population abundance. We assessed the coverage of the CNVR program by estimating the proportion of dogs that were ear-notched and visually scored the health and skin condition of free-roaming dogs in Gelephu and Phuentsholing towns in south Bhutan, bordering India, in September-October 2012. The estimated free-roaming dog population in Gelephu using the Lincoln-Petersen index and Chapman estimates ranged from 612 to 672 and 614 to 671, respectively, while the logit-normal mixed effects model estimate based on the combined two count events was 641 (95% CI: 603-682). In Phuentsholing the Lincoln-Petersen index and Chapman estimates ranged from 525 to 583 and 524 to 582, respectively, while the logit-normal mixed effects model estimate based on the combined four count events was 555 (95% CI: 526-587). The total number of dogs counted was significantly associated with the time of day (AM versus PM; P=0.007), with a 17% improvement in dog sightings during the morning counting events. We recommend to conduct a morning marking followed by one count event the next morning and estimate population size by applying the Lincoln-Peterson corrected Chapman method or conduct two morning count events and apply the logit-normal mixed model to estimate population size. The estimated proportion of vaccinated free-roaming dogs was 56% (95% CI: 52-61%) and 58% (95% CI: 53-62%) in Gelephu and Phuentsholing, respectively. Given coverage in many neighbourhoods was below the recommended threshold of 70%, we recommend conducting an annual "mass dog vaccination only" campaign in southern Bhutan to create an immune buffer in this high rabies-risk area. The male-to-female dog ratio was 1.34:1 in Gelephu and 1.27:1 in Pheuntsholing. Population size estimates using mark-resight surveys has provided useful baseline data for understanding the population dynamics of dogs at the study sites. Mark-resight surveys provide useful information for designing and managing the logistics of dog vaccination or CNVR programs, assessing vaccination coverage, and for evaluating the impact of neutering programs on the size and structure of dog populations over time., (Copyright © 2015. Published by Elsevier B.V.)

The rapid global spread of diseases such as SARS, H5N1, and H1N1 influenza has emphasized the pressing need for trans-disciplinary collaboration and cross-border action, and has also exposed a serious deficit of capacity and coordination in dealing effectively with emerging disease threats. The need for capacity development is particularly acute in the developing world, which is the least well-equipped to respond adequately. Such capacity development can be achieved through education and the implementation of applied 'One Health' activities. This chapter describes the establishment of a 'One Health' capacity development program in South Asia, consisting of two phases. The first phase provides Masters level training for public health doctors and veterinarians, with a focus on epidemiology, and disease control. The second phase reinforces the postgraduate training by establishing a sustainable framework for the implementation of collaborative 'One Health' activities such as the development of multidisciplinary professional networks, implementation of applied zoonotic disease investigation projects, and support for continuing professional development. The objectives are to provide individual skills required to strengthen capacity; to develop an appreciation of the cross-cutting issues which affect human and animal health, set within an institutional context; and to facilitate the development of regional professional networks which will be instrumental in implementing 'One Health' activities.

Aim: The aim of this study is to define the efficacy of dihydrocodeine as an alternative to methadone in the maintenance treatment of opiate dependence., Design: A pragmatic open-label randomized controlled study of patients recommended for opiate maintenance treatment to test equivalence of the two treatment options with follow-up continuing for up to 42 months after recruitment., Setting: Assessment at either Edinburgh's Community Drug Problem Service or at two general practitioner practices with specialist drug community psychiatric nurses, then with shared care follow-up., Participants: Two hundred and thirty-five subjects (168 male, 67 female) with opiate dependence syndrome were recruited. Subjects selected were suitable for opiate maintenance treatment. Routine treatment was offered throughout., Intervention: Patients were randomized to receive either methadone mixture 1 mg/ml or dihydrocodeine, 30 mg or 60 mg tablets., Measurements: The primary outcome measure was retention in treatment. Eight secondary outcomes included total illicit opiate use, reported crime, physical health, mental health, injecting drug use, overdoses, selling drugs and being in education or work. Measures were compared over 42 months follow-up., Findings: There was no difference in groups for retention in treatment at follow-up and there was improvement in all secondary outcomes from baseline. No significant difference in outcomes was found between randomized groups over time. Compliance with randomized treatment differed by randomized group and was affected by experiences in custody during follow-up. Those randomized to dihydrocodeine were more likely to switch treatments., Conclusions: These results, combined with existing clinical experience, provide evidence that dihydrocodeine is a viable alternative to methadone as a maintenance treatment for opiate dependence. Indirect comparisons with other studies show dihydrocodeine (and methadone) to be superior to placebo.

The magnocellular preoptic nucleus of the rat supplies centrifugal input to the olfactory bulb as well as projecting to other olfactory-related areas. The extent to which the piriform and entorhinal cortices can influence the activity of magnocellular preoptic neurons and hence that of the olfactory bulb were examined using intracellular in vivo recording. Stable recordings were obtained in 58 neurons impaled in the magnocellular preoptic nucleus. Antidromic responses occurred on stimulating olfactory bulb (15), piriform cortex (14), or entorhinal area (eight). Monosynaptic excitation was evoked by piriform (27 of 37 tested) and entorhinal cortex (15 of 32 tested) stimulation with polysynaptic inhibition occurring in seven and five neurons, respectively. Polysynaptic as well as antidromic excitation by olfactory bulb stimulation occurred in four; a further 28 tested responded polysynaptically. No response to olfactory bulb stimulation was monosynaptic. In stable impalements, 29 neurons discharged spontaneously in the absence of applied current. Lucifer Yellow and Neurobiotin were used to label 16 cells. All but one had smooth dendrites with soma diameters ranging from 8 to 24 microm. These results provide a framework in which magnocellular preoptic neurons can influence olfactory processing by direct action on the olfactory bulb, which action can be boosted by positive feedback from the bulb through the olfactory piriform and entorhinal cortices.

The magnocellular nucleus (MCPO) was inactivated in anaesthetized rats, using muscimol, a gamma-amino butyric acid ergic agonist, in order to examine the effect of suppression of its tonic activity on extracellular unit firing in the granular (GRL), mitral (MCL) and external plexiform (EPL) layers of the olfactory bulb (OB). In GRL there was a significant increase in unit activity during the first hour after muscimol injection (30 ng), followed by a significant decrease in activity during the following hour. No effect on activity in MCL was seen after muscimol injection into the MCPO. Unit activity in EPL increased during the second hour post-injection. It was concluded that MCPO plays an important part in regulating the balance between granule and tufted cell activity.

The nuclear complex of the horizontal limb of the diagonal band and the magnocellular preoptic nucleus, components of the basal forebrain magnocellular system affected in Alzheimer-type dementia, supply centrifugal innervation to the olfactory bulb. The lateral magnocellular preoptic nucleus provides significant GABAergic input. Since its stimulation may facilitate olfactory bulb mitral cells, we have investigated the effect of sub-total electrolytic lesions in this nucleus on performance in a simple test of olfactory investigation and its habituation. Two groups of rats used with lesions which occupied restricted volumes, approximately 30 and 15% of the magnocellular preoptic nucleus. Behaviorally, there was interference with olfactory investigation, with increased investigation latency and decreased investigation times, the group with larger lesions at 6 and 16 days after operation. There was no significant effect of the smaller lesions. No effects on patterns of olfactory habituation or discrimination were seen. The impairment of olfactory investigation could not be explained by interruption of medial forebrain bundle fibres traversing the nucleus. It is suggested that bilateral partial destruction of magnocellular preoptic neurones may produce significant deficits in either olfactory sensitivity or olfactory motivation.

1. Voltage-gated whole cell Ca2+ currents have been investigated in olfactory bulb (OB) output neurons acutely isolated from neonatal rats. 2. Identification of OB output neurons, mitral or tufted cells, was based on morphology and size and validated by their retrograde labeling with rhodamine or Fast Blue. Of labeled neurons, 45% exhibited either phasic or nonphasic spontaneous firing that was blocked by 10(-7) M tetrodotoxin, 0.5 mM Cd2+, or 1 mM Co2+ in the bathing solution. 3. Whole cell Ca2+ currents displayed holding potential sensitivity indicative of low voltage-activated (LVA) and high voltage-activated (HVA) currents, which exhibited similar dependence on extracellular Ca2+ concentration and could be completely abolished by bathing in 500 microM Cd2+ or in Ca(2+)-free solution. 4. A T-type LVA Ca2+ current, detected in 65% of OB output neurons tested, was activated by depolarizing to -57 mV from holding potential -86 mV and fully inactivated at holding potentials more positive than -60 mV. It was permeated equally by 2.6 mM Ca2+, Sr2+ and Ba2+. The half-activation potential was -35 mV with a slope factor of 7 mV. Depolarizing to -26 mV from different holding potentials in a 2.6-mM Ca2+ solution gave a steady-state half-inactivation potential of -82 mV with a slope factor of 10.7 mV. This LVA current was not sensitive to 5 microM omega-conotoxin (omega-CgTx) or 5 microM Bay K 8644 and was resistant to block by 30 microM Cd2+, by 50 microM verapamil or by 5 microM nifedipine. 5. HVA Ca2+ currents, detected in 97% of OB output cells, activated at around -30 to -20 mV, with maximum peak current at approximately 4 mV in 2.6 mM Ca2+ external solution. They showed similar permeability to 2.6 mM Ca2+ and Sr2+, but the maximum peak current was increased 40% in 2.6 mM Ba2+. Depolarizing to 4 mV from different holding potentials yielded a half-inactivation potential of -67 mV with a slope factor of 13.2 mV. Two components, as suggested by their sensitivities to 5 microM Bay K 8644, nifedipine. omega-CgTx and to voltage, may resemble the L-type and N-type currents described in other neural preparations. However, 5 microM omega-CgTx seemed to block both components, being more effective at more positive potentials. There was a residual component of Cd(2+)-sensitive current not affected by cumulative addition of nifedipine and omega-CgTx. 6. omega-Agatoxin IVA (omega-Aga), a selective P-type Ca2+ channel blocker, had no detectable effect at 50 or 200 nM and 1 microM doses on whole cell Ca2+ currents elicited by 200-ms voltage steps to 4 mV from holding potential -86 mV. 7. We conclude that both LVA and HVA Ca2+ currents exist in neonatal rat OB output neurons, showing distinct kinetic and pharmacological characteristics. The HVA Ca2+ currents contain at least two components, probably resembling L- and N-type currents. Another fast-inactivating HVA component, insensitive to nifedipine, omega-CgTx and omega-Aga, could represent the newly established R-type Ca2+ current.

1. Voltage-gated whole-cell K+ currents have been investigated in olfactory bulb (OB) output (mitral/tufted) neurones from neonatal rats, which were retrogradely labelled by rhodamine or Fast Blue and identified after enzymatic dissociation. Forty-five per cent of labelled neurones exhibited either phasic or non-phasic spontaneous firing in cell-attached configuration. 2. Four outward K+ currents have been identified in all such identified OB output neurones. They are the transient (IA), the delayed rectifier (IDK), and two Ca(2+)-dependent (IK(Ca)) currents. No inward rectifier was detected. 3. The IA was activated at around -45 mV and reached its peak within 3-10 ms. The decay phase could be described by single exponential distribution with the time constant of 45.2 +/- 3.8 ms at depolarizations 10-60 mV from a holding potential of -70 mV. Its activation and steady-state inactivation processes could be fitted with Boltzmann equations yielding half-maximal activation potentials of 7.6 +/- 0.4 and -47.4 +/- 0.2 mV, respectively. It was sensitive to block by 4-AP (1 mM) and by Zn2+ (1 mM). 4. The IDK was activated at potentials more positive than -30 mV, with half-maximal activation at 21 mV. It was sustained during 1 s test pulses without apparent decay. It was blocked by TEA at a concentration of 20 mM. About 8% of the sustained current, in 11/24 cells tested, was found to resist block by a combination of all pharmacological agents tested. 5. Apamin at 100 nM blocked a TEA-insensitive component which accounted for about 23% of the maximal sustained currents. Iberiotoxin (IbTX), which has been found to block maxi K+ currents more selectively than does charybdotoxin, reversibly blocked Ca(2+)-activated K+ current, with a half-maximal dose of about 100 nM in 8/13 OB output neurones tested. This accounted for 20% of the maximal sustained K+ current. The effect of IbTX was not observed in the presence of 20 mM external TEA. 6. Direct evidence is provided in this study regarding kinetic and pharmacological properties of four types of outward K+ channels in OB output neurones.

The nucleus of the horizontal limb of the diagonal band, a component of the basal forebrain magnocellular complex affected in Alzheimer type dementia, supplies centrifugal innervation to the olfactory bulb. We have tested the hypothesis that horizontal limb of the diagonal band lesions will interfere with olfactory memory in a simple olfactory test paradigm. Lesions occupied a restricted volume, approximately 20%, of medial horizontal limb of the diagonal band. There was interference with habituation of investigation latency and duration, six and 16 days after lesioning. It is concluded that bilateral partial lesions of the medial nucleus of the horizontal limb of the diagonal band interfere with habituation memory for odours.

We present intracellular data which demonstrates a unique facilitatory centrifugal influence on the output cells of the olfactory bulb; the source being the lateral component of the nucleus of the horizontal limb of the diagonal band (HDB), part of the basal forebrain magnocellular complex. Damage to this facilitatory HDB influence may explain the loss of olfactory sensitivity seen early in Alzheimer's disease in which pathological changes occur in the basal forebrain.