Mobilized Electrospray Device for On-Tissue Chemical Derivatization in MALDI-MS Imaging.

Applying solutions of a matrix or derivatization agent via microdroplets is a common sample preparation technique for matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) experiments. Mobilized nebulizer sprayers are commonly used to create a homogeneous matrix or reagent layer across large surfaces. Electrospray devices have also been used to produce microdroplets for the same purpose but are rarely used for large tissues due to their immobility. Herein, we present a movable electrospray device that can be used for large tissue sample preparation through a simple modification to an automatic commercial nebulizer device. As demonstrated for on-tissue chemical derivatization (OTCD) with Girard's reagent T using a mimetic tissue model, the sprayer has the additional benefit of being able to investigate reaction acceleration in OTCD when comparing electrostatically charged spray to electrostatically neutral spray. Finally, MALDI-MSI of fatty aldehydes is successfully demonstrated in rat brain tissues using this device for both OTCD and matrix application.

On-tissue chemical derivatization of volatile metabolites for matrix-assisted laser desorption/ionization mass spectrometry imaging.

Mass spectrometry imaging (MSI) of volatile metabolites is challenging, especially in matrix-assisted laser desorption/ionization (MALDI). Most MALDI ion sources operate in vacuum, which leads to the vaporization of volatile metabolites during analysis. In addition, tissue samples are often dried during sample preparation, leading to the loss of volatile metabolites even for other MSI techniques. On-tissue chemical derivatization can dramatically reduce the volatility of analytes. Herein, a derivatization method is proposed utilizing N,N,N-trimethyl-2-(piperazin-1-yl)ethan-1-aminium iodide to chemically modify short-chain fatty acids in chicken cecum, ileum, and jejunum tissue sections before sample preparation for MSI visualization.

Rapid and Automatic Annotation of Multiple On-Tissue Chemical Modifications in Mass Spectrometry Imaging with Metaspace.

On-tissue chemical derivatization is a valuable tool for expanding compound coverage in untargeted metabolomic studies with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). Applying multiple derivatization agents in parallel increases metabolite coverage even further but results in large and more complex datasets that can be challenging to analyze. In this work, we present a pipeline to provide rigorous annotations for on-tissue derivatized MSI data using Metaspace. To test and validate the pipeline, maize roots were used as a model system to obtain MSI datasets after chemical derivatization with four different reagents, Girard's T and P for carbonyl groups, coniferyl aldehyde for primary amines, and 2-picolylamine for carboxylic acids. Using this pipeline helped us annotate 631 unique metabolites from the CornCyc/BraChem database compared to 256 in the underivatized dataset, yet, at the same time, shortening the processing time compared to manual processing and providing robust and systematic scoring and annotation. We have also developed a method to remove false derivatized annotations, which can clean 5-25% of false derivatized annotations from the derivatized data, depending on the reagent. Taken together, our pipeline facilitates the use of broadly targeted spatial metabolomics using multiple derivatization reagents.

Rapid Antibiotic Susceptibility Testing by Deuterium Labeling of Bacterial Lipids in On-Target Microdroplet Cultures.

Antimicrobial resistance is a serious challenge facing human and veterinary health. Current methods of detecting resistance are limited in turn-around time or universal detection. In this work, a new antimicrobial susceptibility test is developed and validated, which utilizes deuterium labeling of membrane lipids to track the growth of bacterial cells. We hypothesize that deuterium uptake and subsequent labeling of lipids can be detected using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Additionally, bacteria growth is performed on the MALDI target, minimizing sample preparation materials and time. When two Escherichia coli strains are grown in the presence of deuterium oxide, labeling can be detected in as little as 30 min to 2 h. The labeling efficiency, or the ratio of labeled to unlabeled lipid peaks, provides information about the growth rate of bacteria. This growth ratio can differentiate between resistant and susceptible strains of bacteria as a resistant strain will maintain ∼50% labeling efficiency between untreated and treated cultures. In comparison, a susceptible strain will see a decrease in fractional abundance of deuterium from ∼50% in the untreated to ∼10% in the treated. This approach is applied to measure the minimum inhibitory concentration (MIC) of the resistant and susceptible strains from on-target microdroplet culture in a range of antibiotic concentrations. The first antibiotic concentration with a significant decrease in fractional abundance of deuterium correlates well with a traditionally obtained MIC using broth dilution, indicating the clinical relevance of the results.

Enhancing Metabolite Coverage for Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Through Multiple On-Tissue Chemical Derivatizations.

The ability to study and visualize metabolites on a cellular and sub-cellular level is important for gaining insights into biological pathways and metabolism of multicellular organisms. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is a powerful analytical tool for metabolomics experiments due to its high sensitivity and small sampling size. The spatial resolution in MALDI-MSI is mainly limited by the number of molecules available in a small sampling size. When the sampling size is low enough to achieve cellular or subcellular spatial resolution, signal intensity is sacrificed making poorly ionized metabolites difficult to detect. To overcome this limitation, on-tissue chemical derivatization reactions have been used to enhance the desorption/ionization efficiency of selected classes of compounds by adding a functional group with a permanent positive charge or one that can be easily ionized. By utilizing several chemical derivatizations in parallel, metabolite coverage can be drastically improved. This chapter outlines methodology for sample preparation and data analysis for on-tissue chemical derivatization using various derivatization reagents.

Corrigendum: Towards Mass Spectrometry Imaging in the Metabolomics Scale: Increasing Metabolic Coverage Through Multiple On-Tissue Chemical Modifications.

[This corrects the article DOI: 10.3389/fpls.2019.00860.].

Toward Mass Spectrometry Imaging in the Metabolomics Scale: Increasing Metabolic Coverage Through Multiple On-Tissue Chemical Modifications.

Exploring the metabolic differences directly on tissues is essential for the comprehensive understanding of how multicellular organisms function. Mass spectrometry imaging (MSI) is an attractive technique toward this goal; however, MSI in metabolomics scale has been hindered by multiple limitations. This is most notable for single cell level high-spatial resolution imaging because of the limited number of molecules in small sampling size and the low ionization yields of many metabolites. Several on-tissue chemical derivatization approaches have been reported to increase MSI signals of targeted compounds, especially in matrix-assisted laser desorption/ionization (MALDI)-MSI. Herein, we adopt a combination of chemical derivatization reactions, to selectively enhance metabolite signals of a specific functional group for each consecutive tissue section. Three well-known on-tissue derivatization methods were used as a proof of concept experiment: coniferyl aldehyde for primary amines, Girard's reagent T for carbonyl groups, and 2-picolylamine for carboxylic acids. This strategy was applied to the cross-sections of leaves and roots from two different maize genotypes (B73 and Mo17), and enabled the detection of over six hundred new unique metabolite features compared to without modification. Statistical analysis indicated quantitative variation between metabolites in the tissue sections, while MS images revealed differences in localization of these metabolites. Combined, this untargeted approach facilitated the visualization of various classes of compounds, demonstrating the potential for untargeted MSI in the metabolomics scale.

Structural Analysis of Polyurethane Monomers by Pyrolysis GC TOFMS via Dopant-Assisted Atmospheric Pressure Chemical Ionization.

Polyurethane is one of the most widely used copolymers and is formed by the cross-linking of isocyanates and polyols. Its physical properties have a strong dependence on the monomer structures, making it very important to characterize the monomers in polyurethane. In this study, we developed a method to analyze unknown polyurethane samples using pyrolysis gas chromatography time-of-flight mass spectrometry (Py-GC-TOFMS) with dopant-assisted atmospheric pressure chemical ionization (dAPCI). A set of standard polyurethane foams produced with several different monomers are analyzed by Py-GC-TOFMS. GC-dAPCI-TOFMS is a high-resolution, soft ionization method for GC-MS analysis that provides accurate mass information of GC separated molecules. The data obtained by this approach could effectively classify different monomers using principal component analysis (PCA), grouping polymers with the same monomers, and providing structural features significant to each monomer. Furthermore, characteristic compounds are identified using in-source collision-induced dissociation (CID) and CSI:FingerID analysis. In contrast, the same set of samples analyzed by Py-GC-electron ionization (EI)-MS could only partially separate some of the monomers. Graphical Abstract .

Does Prophylactic Administration of TXA Reduce Mean Operative Time and Postoperative Blood Loss in Posterior Approach Lumbar Spinal Fusion Surgery Performed for Degenerative Spinal Disease?

STUDY DESIGN: This is a level III retrospective cohort study. OBJECTIVE: To investigate association between prophylactic tranexamic acid (TXA) administration before 1 and 2-level posterior lumbar interbody fusion operations and perioperative blood loss (including intraoperative blood loss and postoperative drain output), mean operative time, postoperative transfusion, and postoperative venous thromboembolic events. SUMMARY OF BACKGROUND DATA: TXA is a systemic antifibrinolytic that competitively inhibits lysine binding sites on plasminogen, reversibly blocking its binding to fibrin and impeding fibrinolysis and clot degradation. TXA's role in routine spinal surgery remains poorly described. Most spinal literature on perioperative TXA administration has considered operations performed for major adult and pediatric spinal deformity. METHODS: Two groups, a study group composed of 75 patients who underwent 1 and 2-level posterior lumbar interbody fusion operations for degenerative indications who received TXA before the start of the procedure, and a control group composed of 75 patients who underwent similar surgeries for the same indications and did not receive TXA preoperatively, were retrospectively enrolled. Demographic, laboratory, and surgical data were collected and analyzed. RESULTS: No statistically significant differences were found between groups with respect to surgery type, home anticoagulation, postoperative anticoagulation, preoperative hemoglobin and hematocrit, estimated intraoperative blood loss, postoperative day 2 drain output, postoperative day 3 drain output, rate of postoperative transfusion, and rate of postoperative thromboembolic events. Statistically significant reductions were noted in the TXA group with regards to postoperative day 1 drain output (P<0.0041), total postoperative drain output (P=0.027), and mean surgical time (P<0.0001). CONCLUSIONS: In the present study, perioperative TXA administration was associated with reduced postoperative drain output and surgical time. Further higher-level studies are required to investigate the safety and utility of TXA's routine use in 1 and 2-level posterior lumbar fusion operations performed for degenerative indications.

Comparison of the Photodegradation of Imazethapyr in Aqueous Solution, on Epicuticular Waxes, and on Intact Corn (Zea Mays) and Soybean (Glycine Max) Leaves.

A direct, controlled comparison of the photodegradation of imazethapyr has been made between imazethapyr in aqueous solutions, imazethapyr on the surface of epicuticular waxes of corn and soybean plants, and imazethapyr on the surface of intact corn and soybean plant leaves. In some experiments, the imazethapyr solutions were allowed to evaporate partially or fully after application to better model environmental conditions. The photodegradation of imazethapyr was fastest in aqueous solutions (k = 0.16 ± 0.02 h-1) and slowest on the surface of corn and soybean plants (kcorn = 0.00048 ± 0.001 h-1 and ksoy = 0.00054 ± 0.003 h-1). Experiments allowing evaporation during irradiation have intermediate rate constants (e.g., kcorn = 0.082 ± 0.005 h-1). Finally, identification of photoproducts was also examined on epicuticular waxes of corn and soybean plants for the first time.

Side-swiped: Ecological cascades emanating from earthworm invasion.

Non-native, invasive earthworms are altering soils throughout the world. Ecological cascades emanating from these changes stem from earthworm-caused changes in detritus processing occurring at a mid-point in the trophic pyramid, rather than the more familiar bottom-up or top-down cascades. They include fundamental changes (microcascades) in soil morphology, bulk density, nutrient leaching, and a shift to warmer, drier soil surfaces with loss of organic horizons. In North American temperate and boreal forests, microcascades cause effects of concern to society (macrocascades), including changes in CO2 sequestration, disturbance regimes, soil quality, water quality, forest productivity, plant communities, and wildlife habitat, and facilitation of other invasive species. Interactions among these changes create cascade complexes that interact with climate change and other environmental changes. The diversity of cascade effects, combined with the vast area invaded by earthworms, lead to regionally important changes in ecological functioning.

Gas Chromatography-Tandem Mass Spectrometry of Lignin Pyrolyzates with Dopant-Assisted Atmospheric Pressure Chemical Ionization and Molecular Structure Search with CSI:FingerID.

Dopant-assisted atmospheric pressure chemical ionization (dAPCI) is a soft ionization method rarely used for gas chromatography-mass spectrometry (GC-MS). The current study combines GC-dAPCI with tandem mass spectrometry (MS/MS) for analysis of a complex mixture such as lignin pyrolysis analysis. To identify the structures of volatile lignin pyrolysis products, collision-induced dissociation (CID) MS/MS using a quadrupole time-of-flight mass spectrometer (QTOFMS) and pseudo MS/MS through in-source collision-induced dissociation (ISCID) using a single stage TOFMS are utilized. To overcome the lack of MS/MS database, Compound Structure Identification (CSI):FingerID is used to interpret CID spectra and predict best matched structures from PubChem library. With this approach, a total of 59 compounds were positively identified in comparison to only 22 in NIST database search of GC-EI-MS dataset. This study demonstrates the effectiveness of GC-dAPCI-MS/MS to overcome the limitations of traditional GC-EI-MS analysis when EI-MS database is not sufficient. Graphical Abstract ᅟ.

TT-TG vs. modified lateral patellar edge for determination of tibial tubercle transfer distance in Fulkerson osteotomy procedures.

BACKGROUND: Tibial tubercle-trochlear groove (TT-TG) distance is currently used at our institution to determine tibial tubercle medialization required in Fulkerson osteotomies. If the correlation between a modified lateral patellar edge (LPE) and the transfer distance was found to be stronger than its correlation with TT-TG, it would suggest that the best measurement to use is actually modified LPE. METHODS: The electronic medical records of 32 patients who underwent Fulkerson osteotomy procedures with femoral nerve stimulation were reviewed and measured. For each patient, modified LPE was measured on quadriceps active hyperextension MRI, and TT-TG was measured on passive extension MRI. Correlation between both TT-TG and LPE and tibial tubercle transfer distance was then determined and analysed. RESULTS: The correlation between TT-TG and actual intra-operative tibial tubercle transfer distance in Fulkerson osteotomy procedures with intra-operative femoral nerve stimulation was found to be weak at 0.436 (p<0.05). The correlation between modified LPE and actual intra-operative tibial tubercle transfer distance was found to be strong at 0.697 (p<0.001). CONCLUSIONS: The correlation between modified LPE and actual intra-operative tibial tubercle transfer distance was stronger than the correlation between TT-TG and tibial tubercle transfer distance. This suggests that the modified LPE may actually be a better preoperative determinant than the currently-utilized TT-TG of the transfer distance required during Fulkerson osteotomy procedures. LEVEL OF EVIDENCE: Level II, diagnostic study.

Correlation of radiographic patellofemoral indices with tibial tubercle transfer distance in Fulkerson osteotomy procedures.

BACKGROUND: A laterally tracking patella is commonly seen in patients with chronic recurrent lateral patellar dislocations. Clinical appearance of the J-sign occurs when the patella is congruent with the trochlear groove in flexion and moves over the lateral border of the femoral condyle as the lower leg reaches complete extension. A Fulkerson osteotomy procedure corrects this maltracking of the patella by medially transferring the tibial tubercle. There are many radiographic patellofemoral indices that can be used describe this incongruence about the patelloformal joint. The current literature supports the use of the tibial tubercle-trochlear groove (TT-TG) index in determining the appropriate amount medialization of the extensor mechanism. However there is little agreement in how far to transfer the tibial tubercle to best achieve maximum patello-femoral congruency. It is the senior author's belief that lateral patellar edge (LPE) measure on voluntary quadriceps active hyperextension MRI scan has the strongest correlation with final operative tibial tubercle transfer distance needed to achieve maximum patellofemoral congruency. PURPOSE: The purpose of this study was to show that the voluntary quadriceps active hyperextension MRI measurement of lateral patellar edge (LPE) has the strongest correlation with tibial tubercle transfer distance required to achieve maxium patellofemoral congruency intraoperatively in the terminal 30 degrees of active knee extension compared to all other patellofemoral indices measured on axial MRI scans with the knee in voluntary active knee extension to 30 degrees of flexion, passive full extension, and voluntary quadriceps active hyperextension. STUDY DESIGN: Retrospective case series via review of the electronic medical record. METHODS: Forty-three Fulkerson osteotomy patient charts were reviewed retrospectively. Three different pre-operative axial MRI views were then examined and measured for Tibial Tubercle-Trochlear Groove (tt-tg), lateral patellar edge (LPE), bisect offset (BSO), and lateral patellar displacement (LPD). Each patient had three MRIs: one with the knee resting in extension, one in voluntary quadriceps active hyperextension, and one in voluntary quadriceps active 30 degree flexion. Statistics were then calculated using Statistical Package for the Social Sciences (SPSS) (IBM corp). RESULTS: Tibial tubercle transfer distances required to achieve congruency intraoperatively correlated moderately (0.500-0.300) and were statistically significant (alpha .050) for passive extension MRI measurement of TT-TG (Pearson--0.403, alpha 0.010) and LPD (Pearson .362, alpha 0.022); voluntary quadriceps active hyperextension TT-TG (Pearson 0.487, alpha, 0.001); voluntary quadriceps active flexion TT-TG (Pearson .548, alpha< 0.001), LPE (Pearson .332, alpha 0.029), and LPD (Pearson 0.446 alpha .003). CONCLUSION: The hypothesis that voluntary quadriceps active hyperextension MRI LPE measurement best correlated with tibial tubercle transfer distance was incorrect. The data collected showed correlation and statistical significance for voluntary quadriceps active flexion LPE with required tibal tubercle transfer distance (Pearson 0.34, alpha 0.026). The MRI measurement that best correlated with tibial tubercle transfer distance was voluntary quadriceps active flexion measure of TT-TG (Pearson .556, alpha< 0.001).

Functional outcomes of MPFL reconstruction vs. graft tissue placement.

BACKGROUND: The medial patellofemoral ligament (MPFL) is essential for the maintenance of correct biomechanical function of the knee. Reconstruction of the MPFL is commonly used in the restoration of patellofemoral stability after traumatic lateral subluxation of the patella. Although a method to accurately determine the MPFL's insertion point has been described, it remains unclear if anatomic placement of MPFL graft tissue is essential for preservation of knee function after MPFL reconstruction. Thus, the purpose of this study was to determine the importance of anatomic placement of MPFL graft tissue for the preservation of knee function following MPFL reconstruction operations. METHODS: Twenty-seven subjects who underwent MPFL reconstruction operations were retrospectively analyzed. Postoperative radiographs were reviewed. Measurements were taken, and the placement of each patient's MPFL graft tissue was determined to be anatomic or non-anatomic based on radiographic methods previously described in the literature. Each subject's electronic medical record was then reviewed, and clinical data was recorded. Finally, the clinical outcomes of each patient were compared to placement location of the MPFL graft tissue in their procedure. RESULTS: Thirteen patients were found to have anatomic MPFL graft tissue placement, and 14 non-anatomic. A significant post-operative difference was found between groups in the following parameters: WOMAC pain (anatomic mean = 85.71 ± 11.34, non-anatomic mean = 75.00 ± 26.35 p = 0.018), function (anatomic mean = 85.85 ± 9.96, non-anatomic mean = 79.09 ± 24.45, p = 0.017) and in KOOS symptom (anatomic mean = 75.63 ± 11.79, non-anatomic mean = 67.83 ± 22.40, p = 0.024), pain (anatomic mean = 77.54 ± 8.61, non-anatomic mean = 71.39 ± 25.18, p = 0.01), ADL (anatomic mean = 85.85 ± 9.97, non-anatomic mean = 79.09 ± 24.45, p = 0.017) and overall (anatomic mean = 74.61 ± 10.33, non-anatomic mean = 69.41 ± 24.25, p = 0.01) scores. No significant difference was observed for post-op instability (p = 0.290) or apprehension (p = 0.496), improvement in WOMAC or KOOS, 2-week, 6-week, or final 1-year range of motion, WOMAC stiffness, or KOOS sport/recreation or QOL. CONCLUSION: Within the range of graft placement values considered by this study, while no reduction in range of motion was seen, non-anatomic placement of MPFL graft tissue in MPFL reconstruction operations caused increased pain and decreased function, evidenced by post-operative KOOS and WOMAC scores. CLINICAL RELEVANCE: It seems that the pivotal step in MPFL reconstruction operations is ensuring correct patellofemoral tracking via intraoperative electrical femoral nerve stimulation. If this step of the procedure is performed correctly, non-anatomic placement will not limit range of motion, lead to continued apprehension, or affect the overall biomechanical functioning of the knee.