Mass Spectrometry Imaging of Arabidopsis thaliana Leaves at the Single-Cell Level by Infrared Laser Ablation Atmospheric Pressure Photoionization (LAAPPI)

In this study, we show that infrared laser ablation atmospheric pressure photoionization mass spectrometry (LAAPPI-MS) imaging with 70 μm lateral resolution allows for the analysis of Arabidopsis thaliana (A. thaliana) leaf substructures ranging from single-cell trichomes and the interveinal leaf lamina to primary, secondary, and tertiary veins. The method also showed its potential for depth profiling analysis for the first time by mapping analytes at the different depths of the leaf and spatially resolving the topmost trichomes and cuticular wax layer from the underlying tissues. Negative ion LAAPPI-MS detected many different flavonol glycosides, fatty acids, fatty acid esters, galactolipids, and glycosphingolipids, whose distributions varied significantly between the different substructures of A. thaliana leaves. The results show that LAAPPI-MS provides a highly promising new tool to study the role of metabolites in plants.Peer reviewe

Soft X-ray Atmospheric Pressure Photoionization in Liquid Chromatography–Mass Spectrometry

Soft X-ray atmospheric pressure photoionization (soft X-ray APPI) as an ionization method in liquid chromatography-mass spectrometry (LC-MS) is presented. The ionization mechanism was examined with selected test compounds in the negative ion mode, using soft X-ray APPI source emitting 4.9 keV photons. Test compounds with an acidic group were ionized by a proton transfer reaction, producing deprotonated molecules ([M-H](-)), whereas compounds having positive electron affinity were ionized by a charge exchange reaction, producing negative molecular ions (M-.). Soft X-ray APPI does not require a dopant to achieve high ionization efficiency, which is an advantage compared with vacuum ultraviolet APPI with 10 eV photons, in which a dopant is needed to improve ionization efficiency. The energy of the soft X-ray photons is in the keV range, which is high enough to displace a valence electron and often also inner shell electrons from LC eluents and atmospheric gases, initiating an efficient ionization process in the negative ion mode.Peer reviewe

Sub-100 mu m Spatial Resolution Ambient Mass Spectrometry Imaging of Rodent Brain with Laser Ablation Atmospheric Pressure Photoionization (LAAPPI) and Laser Ablation Electrospray Ionization (LAESI)

In this study, we applied a new IR laser-beam-focusing technique to enable sub-100 μm spatial resolution in laser ablation atmospheric pressure photoionization (LAAPPI) and laser ablation electrospray ionization (LAESI) mass spectrometry imaging (MSI). After optimization of operational parameters, both LAAPPI- and LAESI-MSI with a spatial resolution of 70 μm produced high-quality MS images, which allowed accurate localization of metabolites and lipids in the mouse and rat brain. Negative and positive ion LAAPPI- and LAESI-MS detected many of the same metabolites and lipids in the brain. Many compounds were also detected either by LAAPPI- or LAESI-MS, indicating that LAAPPI and LAESI are more complementary than alternative methods.In this study, we applied a new IR laser-beamfocusing technique to enable sub-100 mu m spatial resolution in laser ablation atmospheric pressure photoionization (LAAPPI) and laser ablation electrospray ionization (LAESI) mass spectrometry imaging (MSI). After optimization of operational parameters, both LAAPPI- and LAESI-MSI with a spatial resolution of 70 mu m produced high-quality MS images, which allowed accurate localization of metabolites and lipids in the mouse and rat brain. Negative and positive ion LAAPPI- and LAESI-MS detected many of the same metabolites and lipids in the brain. Many compounds were also detected either by LAAPPI- or LAESI-MS, indicating that LAAPPI and LAESI are more complementary than alternative methods.Peer reviewe

Virus-Encapsulated DNA Origami Nanostructures for Cellular Delivery

Peer reviewe

Molecular profile of the rat peri-infarct region four days after stroke: Study with MANF

The peri-infarct region after ischemic stroke is the anatomical location for many of the endogenous recovery processes, and the molecular events in the peri-infarct region remain poorly characterized. In this study, we examine the molecular profile of the peri-infarct region on post-stroke day four, time when reparative processes are ongoing. We used a multiomics approach, involving RNA sequencing, and mass spectrometry-based proteomics and metabolomics to characterize molecular changes in the peri-infarct region. We also took advantage of our previously developed method to express transgenes in the peri-infarct region where self-complementary adeno-associated virus (AAV) vectors were injected into the brain parenchyma on post-stroke day 2. We have previously used this method to show that mesencephalic astrocyte-derived neurotrophic factor (MANF) enhances functional recovery from stroke and recruits phagocytic cells to the peri-infarct region. Here, we first analyzed the effects of stroke to the peri-infarct region on post-stroke day 4 in comparison to sham-operated animals, finding that stroke induced changes in 3345 transcripts, 341 proteins, and 88 metabolites. We found that after stroke genes related to inflammation, proliferation, apoptosis, and regeneration were upregulated, whereas genes encoding neuroactive ligand receptors and calcium-binding proteins were downregulated. In proteomics, we detected upregulation of proteins related to protein synthesis and downregulation of neuronal proteins. Metabolomic studies indicated that in after stroke tissue there is increase in saccharides, sugar phosphates, ceramides and free fatty acids and decrease of adenine, hypoxantine, adenosine and guanosine. We then compared the effects of post-stroke delivery AAV1-MANF delivery to AAV1-eGFP (enhanced green fluorescent protein). MANF administration increased the expression of 77 genes, most of which were related to immune response. In proteomics, MANF administration reduced S100A8 and S100A9 protein levels. In metabolomics, no significant differences between MANF and eGFP treatment were detected, but relative to sham surgery group, most of the changes in lipids were significant in the AAV-eGFP group only. This work describes the molecular profile of the peri-infarct region during recovery from ischemic stroke, and establishes a resource for further stroke studies. These results provide further support for parenchymal MANF as a modulator of phagocytic function.Peer reviewe

BluB/CobT2 fusion enzyme activity reveals mechanisms responsible for production of active form of vitamin B 12 by Propionibacterium freudenreichii

Background Propionibacterium freudenreichii is a food grade bacterium that has gained attention as a producer of appreciable amounts of cobalamin, a cobamide with activity of vitamin B 12 . Production of active form of vitamin is a prerequisite for attempts to naturally fortify foods with B 12 by microbial fermentation. Active vitamin B 12 is distinguished from the pseudovitamin by the presence of 5,6-dimethylbenzimidazole (DMBI) as the lower ligand. Genomic data indicate that P. freudenreichii possesses a fusion gene, bluB/cobT2, coding for a predicted phosphoribosyltransferase/nitroreductase, which is presumably involved in production of vitamin B 12 . Understanding the mechanisms affecting the synthesis of different vitamin forms is useful for rational strain selection and essential for engineering of strains with improved B 12 production properties. Results Here, we investigated the activity of heterologously expressed and purified fusion enzyme BluB/CobT2. Our results show that BluB/CoBT2 is responsible for the biosynthesis of the DMBI base and its activation into α-ribazole phosphate, preparing it for attachment as the lower ligand of cobalamin. The fusion enzyme was found to be efficient in metabolite channeling and the enzymes’ inability to react with adenine, a lower ligand present in the pseudovitamin, revealed a mechanism favoring the production of the active form of the vitamin. P. freudenreichii did not produce cobalamin under strictly anaerobic conditions, confirming the requirement of oxygen for DMBI synthesis. In vivo experiments also revealed a clear preference for incorporating DMBI over adenine into cobamide under both microaerobic and anaerobic conditions. Conclusions The herein described BluB/CobT2 is responsible for the production and activation of DMBI. Fusing those two activities results in high pressure towards production of the true vitamin B 12 by efficiently activating DMBI formed within the same enzymatic complex. This indicates that BluB/CobT2 is the crucial enzyme in the B 12 biosynthetic pathway of P. freudenreichii. The GRAS organism status and the preference for synthesizing active vitamin form make P. freudenreichii a unique candidate for the in situ production of vitamin B 12 within food products. Keywords: Propionibacterium freudenreichii ; Cobalamin; B12; DMBI; α-Ribazole; Phosphoribozyltransferase; Nitroreductase; Fusion enzyme; BluB; CobTPeer reviewe

Adrenocortical carcinoma: presentation and outcome of a contemporary patient series

Background: Adrenocortical carcinoma (ACC) is a rare endocrine carcinoma with poor 5-year survival rates of Design and methods: We studied all patients (n = 47, four children) from a single centre during years 2002–2018. We re-evaluated radiologic and histopathological findings and assessed treatments and outcome. We searched for possible TP53 gene defects and assessed nationwide incidence of ACC.Results: In adults, incidental radiologic finding led to diagnosis in 79% at median age of 61 years. ENSAT stage I, II, III and IV was 19%, 40%, 19% and 21%, respectively. Nonenhanced CT demonstrated > 20 Hounsfield Units (HU) for all tumours (median 34 (21–45)), median size 92 mm (20–196), Ki67 17% (1–40%), Weiss score 7 (4–9) and Helsinki score 24 (4–48). ACC was more often found in the left than the right adrenal (p  5 to > 10 years was achieved after repeated surgery of metastases. Overall 5-year survival was 67%, and 96% vs. 26% for ENSAT stage I–II vs. III–IV (p Conclusions: Contemporary ACC predominantly presents as an incidental imaging finding, characterised by HU > 20 on nonenhanced CT but variable tumour size (20–196 mm). Malignancy cannot be ruled out by small tumour size only. The 5-year survival of 96% in ENSAT stage I–III compares favourably to previous studies.</p

Clinical assessment of a non-invasive wearable MEMS pressure sensor array for monitoring of arterial pulse waveform, heart rate and detection of atrial fibrillation

There is an unmet clinical need for a low cost and easy to use wearable devices for continuous cardiovascular health monitoring. A flexible and wearable wristband, based on microelectromechanical sensor (MEMS) elements array was developed to support this need. The performance of the device in cardiovascular monitoring was investigated by (i) comparing the arterial pressure waveform recordings to the gold standard, invasive catheter recording (n = 18), (ii) analyzing the ability to detect irregularities of the rhythm (n = 7), and (iii) measuring the heartrate monitoring accuracy (n = 31). Arterial waveforms carry important physiological information and the comparison study revealed that the recordings made with the wearable device and with the gold standard device resulted in almost identical (r = 0.9-0.99) pulse waveforms. The device can measure the heart rhythm and possible irregularities in it. A clustering analysis demonstrates a perfect classification accuracy between atrial fibrillation (AF) and sinus rhythm. The heartrate monitoring study showed near perfect beat-to-beat accuracy (sensitivity = 99.1%, precision = 100%) on healthy subjects. In contrast, beat-to-beat detection from coronary artery disease patients was challenging, but the averaged heartrate was extracted successfully (95% CI: -1.2 to 1.1 bpm). In conclusion, the results indicate that the device could be useful in remote monitoring of cardiovascular diseases and personalized medicine