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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.

Oral squamous cell carcinoma (OSCC) is the prevalent type of oral cavity cancer, requiring precise, accurate, and affordable diagnosis to identify the disease in early stages, Comprehending the differences in lipid profiles between healthy and cancerous tissues encompasses great relevance in identifying biomarker candidates and enhancing the odds of successful cancer treatment. Therefore, the present study evaluates the analytical performance of simultaneous mRNA and lipid extraction in gingiva tissue from healthy patients and patients diagnosed with OSCC preserved in TRIzol reagent. The data was analyzed by partial least-squares discriminant analysis (PLS-DA) and confirmed via matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). The lipid extraction in TRIzol solution was linear in a range from 330 to 2000 ng mL-1, r2 > 0.99, intra and interday precision and accuracy <15%, and absolute recovery values ranging from 90 to 110%. The most important lipids for tumor classification were evaluated by MALDI-MSI, revealing that the lipids responsible for distinguishing the OSCC group are more prevalent in the cancerous tissue in contrast to the healthy group. The results exhibit the possibilities to do transcriptomic and lipidomic analyses in the same sample and point out important candidates related to the presence of OSCC.

Grafting is widely used in horticulture. Shortly after grafting, callus tissues appear at the graft interface and the vascular tissues of the scion and rootstock connect. The graft interface contains a complex mix of tissues, we hypothesised that each tissue has its own metabolic response to wounding/grafting and accumulates different metabolites at different rates. We made intact and wounded cuttings and grafts of grapevine, and then measured changes in bulk flavonoid, phenolic acid and stilbenoid concentration and used metabolite imaging to study tissue-specific responses. We show that some metabolites rapidly accumulate in specific tissues after grafting, for example, stilbene monomers accumulate in necrotic tissues surrounding mature xylem vessels. Whereas other metabolites, such as complex stilbenes, accumulate in the same tissues at later stages. We also observe that other metabolites accumulate in the newly formed callus tissue and identify genotype-specific responses. In addition, exogenous resveratrol application did not modify grafting success rate, potentially suggesting that the accumulation of resveratrol at the graft interface is not linked to graft union formation. The increasing concentration of complex stilbenes often occurs in response to plant stresses (via unknown mechanisms), and potentially increases antioxidant activity and antifungal capacities.

Bioaccumulation of organic contaminants from contaminated food sources might pose an underestimated risk toward shredding invertebrates. This assumption is substantiated by monitoring studies observing discrepancies of predicted tissue concentrations determined from laboratory-based experiments compared with measured concentrations of systemic pesticides in gammarids. To elucidate the role of dietary uptake in bioaccumulation, gammarids were exposed to leaf material from trees treated with a systemic fungicide mixture (azoxystrobin, cyprodinil, fluopyram, and tebuconazole), simulating leaves entering surface waters in autumn. Leaf concentrations, spatial distribution, and leaching behavior of fungicides were characterized using liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-HRMS/MS) and matrix-assisted laser desorption ionization-mass spectrometric imaging. The contribution of leached fungicides and fungicides taken up from feeding was assessed by assembling caged (no access) and uncaged (access to leaves) gammarids. The fungicide dynamics in the test system were analyzed using LC-HRMS/MS and toxicokinetic modeling. In addition, a summer scenario was simulated where water was the initial source of contamination and leaves contaminated by sorption. The uptake, translocation, and biotransformation of systemic fungicides by trees were compound-dependent. Internal fungicide concentrations of gammarids with access to leaves were much higher than in caged gammarids of the autumn scenario, but the difference was minimal in the summer scenario. In food choice and dissectioning experiments gammarids did not avoid contaminated leaves and efficiently assimilated contaminants from leaves, indicating the relevance of this exposure pathway in the field. The present study demonstrates the potential impact of dietary uptake on in situ bioaccumulation for shredders in autumn, outside the main application period. The toxicokinetic parameters obtain

Abstract: In wet-milling of maize, an approach to obtain a more efficient extraction of starch is to use enzymes to degrade cell-wall polysaccharides that encapsulates the starch granules. To enhance the understanding of how these enzymes function in the maize kernel, mass spectrometry imaging was used to compare the oligosaccharide degradation products formed and their distribution in maize kernels after treatment with three xylanases. For the measurements, amylase treated cryosections of maize kernels were sprayed with different xylanases on approx. one half of the surface area and then incubated for 17 h. Mass spectrometry images were then generated using an atmospheric pressure MALDI-MSI system. The images show the distributions of a wide variety of oligosaccharides and the enzyme ability to produce these oligosaccharides in distinct locations of the maize kernels. The approach provides new information on how the efficiency of xylanase to form specific degradations products are dependent on the morphological regions of maize kernels. It also provides indirect information about possible changes in the constituents of the arabinoxylan substrate by showing the distributions of oligosaccharides with various decorations of the xylan backbone.

Quality of life is often reduced in patients with sleep-wake disorders. Insomnia is commonly treated with benzodiazepines, despite their well-known side effects. Pellotine (1), a Lophophora alkaloid, has been reported to have short-acting sleep-inducing properties in humans. In this study, we set out to evaluate various in vitro and in vivo properties of 1. We demonstrate that 1 undergoes slow metabolism; e.g. in mouse liver microsomes 65% remained, and in human liver microsomes virtually no metabolism was observed after 4 h. In mouse liver microsomes, two phase I metabolites were identified: 7-desmethylpellotine and pellotine-N-oxide. In mice, the two diastereomers of pellotine-O-glucuronide were additionally identified as phase II metabolites. Furthermore, we demonstrated by DESI-MSI that 1 readily enters the central nervous system of rodents. Furthermore, radioligand-displacement assays showed that 1 is selective for the serotonergic system and in particular the serotonin (5-HT)1D, 5-HT6, and 5-HT7 receptors, where it binds with affinities in the nanomolar range (117, 170, and 394 nM, respectively). Additionally, 1 was functionally characterized at 5-HT6 and 5-HT7, where it was found to be an agonist at the former (EC50 = 94 nM, Emax = 32%) and an inverse agonist at the latter (EC50 = 291 nM, Emax = -98.6). Finally, we demonstrated that 1 dose-dependently decreases locomotion in mice, inhibits REM sleep, and promotes sleep fragmentation. Thus, we suggest that pellotine itself, and not an active metabolite, is responsible for the hypnotic effects and that these effects are possibly mediated through modulation of serotonergic receptors.

Background Patients with hypertrophic scars (HTS) risk reduced quality of life due to itching, pain, poor cosmesis, and restriction of movement. Despite good clinical efficacy, patients are often reluctant to undergo repeated needle injections due to pain or needle phobia. Objectives To evaluate the applicability of needle-free pneumatic jet injection (PJI) and assess changes in hypertrophic scars following a single PJI treatment with 5-fluorouracil (5-FU) and triamcinolone acetonide (TAC). Methods Twenty patients completed this blinded, randomized, controlled, split-scar trial. The intervention side of the HTS received a one-time treatment with PJIs containing a mixture of TAC + 5-FU injected at 5 mm intervals (mean 7 PJI per HTS); the control side received no treatment. Assessments were made at baseline and 4 weeks posttreatment. Outcome measures included change in (1) Vancouver Scar Scale (VSS) total score and subscores, (2) scar volume and surface area assessed by three-dimensional imaging, (3) skin microarchitecture measured by optical-coherence tomography (OCT), (4) photo-assessed scar cosmesis (0-100), (5) patient-reported pain and satisfaction (0-10), and (6) depiction of drug biodistribution after PJI. Results PJI with TAC + 5-FU significantly decreased both HTS height (-1 VSS; p = 0.01) and pliability (-1 VSS; p < 0.01) with a nonstatistically significant reduction of -1 in total VSS score (0 in control; p = 0.09). On 3D imaging, a 33% decrease in scar volume (p = 0.016) and a 37% decrease in surface area (p = 0.008) was observed. OCT indicated trends towards smoother scar surface (Ra 11.1-10.3; p = 0.61), normalized dermal microarchitecture (attenuation coefficient: 1.52-1.68; p = 0.44), and a reduction in blood flow between 9% and 17% (p = 0.50-0.79). Despite advances in VSS subscores and OCT, no improved photo-assessed cosmesis was found (-3.2 treatment vs. -1.4 control; p = 0.265). Patient-reported pain was low (2/10) and 90% of the patients that

Background: Intralesional bleomycin (BLM) administration by needle injection is effective for keloids and warts but has significant drawbacks, including treatment-related pain and operator-depended success rates. Electronic pneumatic injection (EPI) is a promising, less painful, needle-free method that potentially enables precise and controlled dermal drug delivery. Here, we aimed to explore the cutaneous pharmacokinetics, biodistribution patterns, and tolerability of BLM administered by EPI in vivo. Research Design and Methods: In a pig model, EPI with BLM or saline (SAL) were evaluated after 1, 48 and 216 hours. Mass spectrometry quantification and imaging were used to assess BLM concentrations and biodistribution patterns in skin biopsies. Tolerability was assessed by scoring local skin reactions (LSR) and measuring transepidermal water loss (TEWL). Results: Directly after BLM injection a peak concentration of 109.2 µg/cm3 (43.9–175.2) was measured in skin biopsies. After 9 days BLM was undetectable. EPI resulted in a focal BLM biodistribution in the mid-dermal delivery zone resembling a triangular shape. Mild LSRs were resolved spontaneously and TEWL was unaffected. Conclusions: BLM administered by EPI resulted in quantifiable and focal mid-dermal distribution of BLM. The high skin bioavailability holds a great potential for clinical effects and warrants further evaluation in future human studies.

The aim of Quantitative mass spectrometry imaging (Q-MSI) is to provide distribution analysis and quantitation from one single mass-spectrometry-based experiment, and several quantitation methods have been devised for Q-MSI. Mimetic tissue models based on spiked tissue homogenates are considered one of the most accurate ways to perform Q-MSI, since the analyte is present in a well-defined concentration in a sample matrix highly similar to the one of the unknown sample to be analyzed. The delivery of drugs in skin is among the most frequent types of pharmaceutical MSI studies. Here, a mimetic tissue model is extended for use on the skin, which, due to its high collagen content, is different from most other tissue as the homogenates become extremely viscous. A protocol is presented which overcomes this by the addition of water and the handling of the homogenate at an elevated temperature where the viscosity is lower. Using a mimetic tissue model, a method was developed for the quantitative imaging of bleomycin in skin. To compensate for the signal drift and the inhomogeneities in the skin, an internal standard was included in the method. The method was tested on skin from a pig which had had an electropneumatic injection of bleomycin into the skin. Quantification was made at several regions in a cross section of the skin at the injection site, and the results were compared to the results of a quantitative LC-MS on a neighboring tissue biopsy from the same animal experiment. The overall tissue concentration determined by the LC-MS was within the range of the different regions quantified by the Q-MSI. As the model provides the results of the same order of magnitude as a LC-MS, it can either be used to replace LC-MS in skin studies where MSI and LC-MS are today carried out in combination, or it can add quantitative information to skin studies which are otherwise carried out by MSI alone.

New mass spectrometry imaging (MSI) techniques are gaining importance in various stages of the drug discovery and development chain as well as in more fundamental studies of drug delivery. The advantage of MSI is that for an imaging technique, the selectivity is very high and as a label-free technique, it enables simultaneous and compound-specific imaging of drugs and their metabolites. In addition to that, a vast number of endogenous metabolites, which may serve as tissue biomarkers, can be imaged simultaneously, providing a very extensive characterization of the sample. The present review focusses on the application of MSI for imaging of drugs and their metabolites, primarily is studies of drug distribution and drug metabolism. The first half provides a quite general introduction to MSI with the main focus on MALDI-MSI and DESI-MSI, which are the two most commonly applied MSI techniques for pharmaceutical applications. It also has sections where the necessary sample preparation, including cryo-sectioning, is discussed, along with sections on data analysis and quantitative MSI, which in particular for pharmaceutical applications plays an increasingly significant role. The second half of the paper presents examples of pharmaceutical applications of MSI. The chapter does not contain an exhaustive list of all published studies, but should be considered a curated list of examples where MSI has contributed with information which cannot readily be obtained by conventional analytical techniques. The examples are chosen to represent diverse fields within drug delivery (local and systemic) and drug metabolism with examples from different organs and from whole-body studies. The final section presents a broader look at ongoing trends within MSI and some thoughts on where MSI is heading in the years to come.

Background: Opening of KATP channels by systemic levcromakalim treatment triggers attacks in migraine patients and hypersensitivity to von Frey stimulation in a mouse model. Blocking of these channels is effective in several preclinical migraine models. It is unknown in what tissue and cell type KATP-induced migraine attacks are initiated and which KATP channel subtype is targeted. Methods: In mouse models, we administered levcromakalim intracerebroventricularly, intraperitoneally and intraplantarily and compared the nociceptive responses by von Frey and hotplate tests. Mice with a conditional loss-of-function mutation in the smooth muscle KATP channel subunit Kir6.1 were given levcromakalim and GTN and examined with von Frey filaments. Arteries were tested for their ability to dilate ex vivo. mRNA expression, western blotting and immunohistochemical stainings were made to identify relevant target tissue for migraine induced by KATP channel opening. Results: Systemic administration of levcromakalim induced hypersensitivity but central and local administration provided antinociception respectively no effect. The Kir6.1 smooth muscle knockout mouse was protected from both GTN and levcromakalim induced hypersensitivity, and their arteries had impaired dilatory response to the latter. mRNA and protein expression studies showed that trigeminal ganglia did not have significant KATP channel expression of any subtype, whereas brain arteries and dura mater primarily expressed the Kir6.1 + SUR2B subtype. Conclusion: Hypersensitivity provoked by GTN and levcromakalim in mice is dependent on functional smooth muscle KATP channels of extracerebral origin. These results suggest a vascular contribution to hypersensitivity induced by migraine triggers.