Multimodal imaging of biological tissues using combined MALDI and NAPA-LDI mass spectrometry for enhanced molecular coverage.

Mass spectrometry imaging (MSI) is a powerful analytical technique that enables detection, discovery, and identification of multiple classes of biomolecules, while simultaneously mapping their spatial distributions within a sample (e.g., a section of biological tissue). The limitation in molecular coverage afforded by any single MSI platform has led to the development of multimodal approaches that incorporate two or more techniques to obtain greater chemical information. Matrix-assisted laser desorption ionization (MALDI) is a preeminent ionization technique for MSI applications because the wide range of available matrices allows some degree of enhancement with respect to the detection of particular molecular classes. Nonetheless, MALDI has a limited ability to detect and image several classes of molecules, e.g., neutral lipids, in complex samples. Laser desorption ionization from silicon nanopost arrays (NAPA-LDI or NAPA) has been shown to offer complementary coverage with respect to MALDI by providing improved detection of neutral lipids and some small metabolites. Here, we present a multimodal imaging method in which a single tissue section is consecutively imaged at low and high laser fluences, generating spectra that are characteristic of MALDI and NAPA ionization, respectively. The method is demonstrated to map the distributions of species amenable to detection by MALDI (e.g., phospholipids and intermediate-mass metabolites) and NAPA (e.g., neutral lipids such as triglycerides and hexosylceramides, and small metabolites) in mouse brain and lung tissue sections.

Remote ablation chamber for high efficiency particle transfer in laser ablation electrospray ionization mass spectrometry.

Laser ablation electrospray ionization (LAESI) driven by mid-infrared laser pulses allows the direct analysis of biological tissues with minimal sample preparation. Dedicated remote ablation chambers have been developed to eliminate the need for close proximity between the sample and the mass spectrometer inlet. This also allows for the analysis of large or irregularly shaped objects, and incorporation of additional optics for microscopic imaging. Here we report on the characterization of a newly designed conical inner volume ablation chamber working in transmission geometry, where a reduced zone of stagnation was achieved by tapering the sample platform and the chamber outlet. As a result, the transmission efficiency of both large (>7.5 µm) and smaller particulates (<6.5 µm) has increased significantly. Improved analytical figures of merit, including 300 fmol limit of detection, and three orders of magnitude in dynamic range, were established. Particle residence time, measured by the FWHM of the analyte signal, was reduced from 2.0 s to 0.5 s enabling higher ablation rates and shorter analysis time. A total of six glucosinolates (sinigrin, gluconapin, progoitrin, glucoiberin, glucoraphanin, and glucohirsutin) were detected in plant samples with ion abundances higher by a factor of 2 to 8 for the redesigned ablation chamber.

High Throughput Complementary Analysis and Quantitation of Metabolites by MALDI- and Silicon Nanopost Array-Laser Desorption/Ionization-Mass Spectrometry.

Silicon nanopost array (NAPA) structures have been shown to be effective substrates for laser desorption/ionization-mass spectrometry (LDI-MS) and have been used to analyze a variety of samples including peptides, metabolites, drugs, explosives, and intact cells, as well as to image lipids and metabolites in tissue sections. However, no direct comparison has yet been conducted between NAPA-MS and the most commonly used LDI-MS technique, matrix-assisted laser desorption/ionization (MALDI)-MS. In this work, we compare the utility of NAPA-MS to that of MALDI-MS using two common matrices for the analysis of metabolites in cellular extracts and human urine. Considerable complementarity of molecular coverage was observed between the two techniques. Of 178 total metabolites assigned from cellular extracts, 68 were uniquely detected by NAPA-MS and 62 were uniquely detected by MALDI-MS. NAPA-MS was found to provide enhanced coverage of low-molecular weight compounds such as amino acids, whereas MALDI afforded better detection of larger, labile compounds including nucleotides. In the case of urine, a sample largely devoid of higher-mass labile compounds, 88 compounds were uniquely detected by NAPA-MS and 13 by MALDI-MS. NAPA-MS also favored more extensive alkali metal cation adduction relative to MALDI-MS, with the [M + 2Na/K - H]+ species accounting for as much as 97% of the total metabolite ion signal in positive mode. The capability of NAPA-MS for targeted quantitation of endogenous metabolites in urine via addition of isotopically labeled standards was also examined. Both NAPA-MS and MALDI-MS provided quantitative results in good agreement with one another and the concentrations reported in the literature, as well as good sample-to-sample reproducibility (RSD < 10%).

Enhanced sensitivity and metabolite coverage with remote laser ablation electrospray ionization-mass spectrometry aided by coaxial plume and gas dynamics.

Laser ablation electrospray ionization-mass spectrometry (LAESI-MS) allows for direct analysis of biological tissues at atmospheric pressure with minimal to no sample preparation. In LAESI, a mid-IR laser beam (λ = 2.94 µm) is focused onto the sample to produce an ablation plume that is intercepted and ionized by an electrospray at the inlet of the mass spectrometer. In the remote LAESI platform, the ablation process is removed from the mass spectrometer inlet and takes place in an ablation chamber, allowing for incorporation of additional optics for microscopic imaging and targeting of specific features of the sample for laser ablation sampling. The ablated material is transported by a carrier gas through a length of tubing, delivering it to the MS inlet where it is intercepted and ionized by an electrospray. Previous proof-of-principle studies used a prolate spheroid ablation chamber with the carrier gas flow perpendicular to the ablation plume. This design resulted in significant losses of MS signal in comparison to conventional LAESI. Here we present a newly designed conical inner volume ablation chamber that radially confines the ablation plume produced in transmission geometry. The carrier gas flow and the expanding ablation plume are aligned in a coaxial configuration to improve the transfer of ablated particles. This new design not only recovered the losses observed with the prolate spheroid chamber design, but was found to provide an ∼12-15% increase in the number of metabolite peaks detected from plant leaves and tissue sections relative to conventional LAESI.

Molecular Imaging of Biological Samples on Nanophotonic Laser Desorption Ionization Platforms.

Mass spectrometry imaging (MSI) is a comprehensive tool for the analysis of a wide range of biomolecules. The mainstream method for molecular MSI is matrix-assisted laser desorption ionization, however, the presence of a matrix results in spectral interferences and the suppression of some analyte ions. Herein we demonstrate a new matrix-free MSI technique using nanophotonic ionization based on laser desorption ionization (LDI) from a highly uniform silicon nanopost array (NAPA). In mouse brain and kidney tissue sections, the distributions of over 80 putatively annotated molecular species are determined with 40 µm spatial resolution. Furthermore, NAPA-LDI-MS is used to selectively analyze metabolites and lipids from sparsely distributed algal cells and the lamellipodia of human hepatocytes. Our results open the door for matrix-free MSI of tissue sections and small cell populations by nanophotonic ionization.

HSP70 natively and specifically associates with an N-terminal dermcidin-derived peptide that contains an HLA-A*03 antigenic epitope.

Tumor cells very often have elevated expression of HSP70, the anti-apoptotic properties of which contribute to overall tumor survival. Independent of its anti-apoptotic properties, HSP70 was also suggested to be involved in the antigen presentation process by chaperoning cytosolic peptides, thus protecting them from rapid degradation and securing the peptide pool for further processing. In this study, we identified a 33-amino acid N-terminal dermcidin (DCD)-derived peptide from the repertoire of in vivo HSP70-associated peptides isolated from a leukemic cell line, K562. The DCD peptide has been previously shown to be involved in tumorigenesis, to increase tumor survival rate, to improve tumor stress resistance, and to aid growth. We show that HSP70 is a specific binding partner for the DCD prosurvival peptide and define an ATP-dependent DCD-binding site (GNPCH). We also identify an HLA-A*03 antigenic epitope within the DCD peptide, which follows and partially overlaps the HSP70-binding site (CHEASAAQK). This study describes the interaction between HSP70 and the DCD-derived prosurvival peptide, an interaction that may direct the peptide toward antigen presentation and independently contribute to the prosurvival mechanism mediated by DCD.

Coral mucus: the properties of its constituent mucins.

The gel-forming properties of mucus are closely related to its functioning; although there is limited information available relating to coral mucus gels. The present study investigates coral mucus glycoprotein using rheological methods. We demonstrate the presence of a high-molecular-weight polymeric glycoprotein similar to that found in vertebrates, capable of forming a gel. The milked mucus exuded mostly from the oral cavity of corals is not a gel; however, it does show a tendency to form a gel upon concentration. Such results indicate the potential for corals to produce two different kinds of mucus, each potentially capable of performing different functions.

Graphene-based encapsulation of liquid metal particles.

Liquid metals are a promising functional material due to their unique combination of metallic properties and fluidity at room temperature. They are of interest in wide-ranging fields including stretchable and flexible electronics, reconfigurable devices, microfluidics, biomedicine, material synthesis, and catalysis. Transformation of bulk liquid metal into particles has enabled further advances by allowing access to a broader palette of fabrication techniques for device manufacture or by increasing area available for surface-based applications. For gallium-based liquid metal alloys, particle stabilization is typically achieved by the oxide that forms spontaneously on the surface, even when only trace amounts of oxygen are present. The utility of the particles formed is governed by the chemical, electrical, and mechanical properties of this oxide. To overcome some of the intrinsic limitations of the native oxide, it is demonstrated here for the first time that 2D graphene-based materials can encapsulate liquid metal particles during fabrication and imbue them with previously unattainable properties. This outer encapsulation layer is used to physically stabilize particles in a broad range of pH environments, modify the particles' mechanical behavior, and control the electrical behavior of resulting films. This demonstration of graphene-based encapsulation of liquid metal particles represents a first foray into the creation of a suite of hybridized 2D material coated liquid metal particles.

Mass spectrometry imaging of triglycerides in biological tissues by laser desorption ionization from silicon nanopost arrays.

Mass spectrometry imaging (MSI) is used increasingly to simultaneously detect a broad range of biomolecules while mapping their spatial distributions within biological tissue sections. Matrix-assisted laser desorption ionization (MALDI) is recognized as the method-of-choice for MSI applications due in part to its broad molecular coverage. In spite of the remarkable advantages offered by MALDI, imaging of neutral lipids, such as triglycerides (TGs), from tissue has remained a significant challenge due to ion suppression of TGs by phospholipids, e.g. phosphatidylcholines (PCs). To help overcome this limitation, silicon nanopost array (NAPA) substrates were introduced to selectively ionize TGs from biological tissue sections. This matrix-free laser desorption ionization (LDI) platform was previously shown to provide enhanced ionization of certain lipid classes, such as hexosylceramides (HexCers) and phosphatidylethanolamines (PEs) from mouse brain tissue. In this work, we present NAPA as an MSI platform offering enhanced ionization efficiency for TGs from biological tissues relative to MALDI, allowing it to serve as a complement to MALDI-MSI. Analysis of a standard lipid mixture containing PC(18:1/18:1) and TG(16:0/16:0/16:0) by LDI from NAPA provided an ~49 and ~227-fold higher signal for TG(16:0/16:0/16:0) relative to MALDI, when analyzed without and with the addition of a sodium acetate, respectively. In contrast, MALDI provided an ~757 and ~295-fold higher signal for PC(18:1/18:1) compared with NAPA, without and with additional Na+ . Averaged signal intensities for TGs from MSI of mouse lung and human skin tissues exhibited an ~105 and ~49-fold increase, respectively, with LDI from NAPA compared with MALDI. With respect to PCs, MALDI provided an ~2 and ~19-fold increase in signal intensity for mouse lung and human skin tissues, respectively, when compared with NAPA. The complementary coverage obtained by the two platforms demonstrates the utility of using both techniques to maximize the information obtained from lipid MS or MSI experiments.

Chemically modifying the mechanical properties of core-shell liquid metal nanoparticles.

Eutectic gallium-indium (EGaIn) is a room temperature liquid metal that can be readily fabricated into nanoparticles which naturally form a thin, passivating gallium oxide shell. These core-shell nanoparticles are of interest for a variety of stimuli-responsive applications, which often utilize physical deformation of the particles to release the molten, conductive payload from within the gallium oxide shell. In the present work, we introduce a variety of chemical strategies to produce EGaIn nanoparticles which exhibit a wide range of gallium oxide shell thicknesses. These chemically modified oxide thicknesses are then correlated to the core-shell liquid nanoparticles' mechanical properties by subjecting the particles to orthogonal characterization techniques; XPS for measurement of the gallium oxide shell thickness and nanoindentation for measurement of particle stiffness and elastic modulus. Additionally, nanoindentation is used to determine the onset of particle rupture and resultant conductivity. Ultimately, quantification of the relationships between chemical treatment and derivative mechanical properties in liquid metal nanoparticles will enable advanced applications of these colloids, such as in tailored self-healing and responsive electronic devices.

Mechanoresponsive Polymerized Liquid Metal Networks.

Room-temperature liquid metals, such as nontoxic gallium alloys, show enormous promise to revolutionize stretchable electronics for next-generation soft robotic, e-skin, and wearable technologies. Core-shell particles of liquid metal with surface-bound acrylate ligands are synthesized and polymerized together to create cross-linked particle networks comprising >99.9% liquid metal by weight. When stretched, particles within these polymerized liquid metal networks (Poly-LMNs) rupture and release their liquid metal payload, resulting in a rapid 108 -fold increase in the network's conductivity. These networks autonomously form hierarchical structures that mitigate the deleterious effects of strain on electronic performance and give rise to emergent properties. Notable characteristics include nearly constant resistances over large strains, electronic strain memory, and increasing volumetric conductivity with strain to over 20 000 S cm-1 at >700% elongation. Furthermore, these Poly-LMNs exhibit exceptional performance as stretchable heaters, retaining 96% of their areal power across relevant physiological strains. Remarkable electromechanical properties, responsive behaviors, and facile processing make Poly-LMNs ideal for stretchable power delivery, sensing, and circuitry.

Matrix-free mass spectrometry imaging of mouse brain tissue sections on silicon nanopost arrays.

Mass spectrometry imaging (MSI) is capable of detection and identification of diverse classes of compounds in brain tissue sections, whereas simultaneously mapping their spatial distributions. Given the vast array of chemical components present in neurological systems, as well as the innate diversity within molecular classes, MSI platforms capable of detecting a wide array of species are useful for achieving a more comprehensive understanding of their biological roles and significance. Currently, matrix-assisted laser desorption ionization (MALDI) is the method of choice for the molecular imaging of brain samples by mass spectrometry. However, nanostructured laser desorption ionization platforms, such as silicon nanopost arrays (NAPA), are emerging as alternative MSI techniques that can provide complementary insight into molecular distributions in the central nervous system. In this work, the molecular coverage of mouse brain lipids afforded by NAPA-MSI is compared to that of MALDI-MSI using two common MALDI matrices. In positive ion mode, MALDI spectra were dominated by phosphatidylcholines and phosphatidic acids. NAPA favored the ionization of phosphatidylethanolamines and glycosylated ceramides, which were poorly detected in MALDI-MSI. In negative ion mode, MALDI favored sulfatides and free fatty acids, whereas NAPA spectra were dominated by signal from phosphatidylethanolamines. The complementarity in lipid coverages between the NAPA- and MALDI-MSI platforms presents the possibility of selective lipid analysis and imaging dependent upon which platform is used. Nanofabrication of the NAPA platform offers better uniformity compared to MALDI, and the wider dynamic range offered by NAPA promises improved quantitation in imaging.

Mass Spectrometry Imaging of Lipids in Human Skin Disease Model Hidradenitis Suppurativa by Laser Desorption Ionization from Silicon Nanopost Arrays.

Neutral lipids have been implicated in a host of potentially debilitating human diseases, such as heart disease, type-2 diabetes, and metabolic syndrome. Matrix-assisted laser desorption ionization (MALDI), the method-of-choice for mass spectrometry imaging (MSI), has led to remarkable success in imaging several lipid classes from biological tissue sections. However, due to ion suppression by phospholipids, MALDI has limited ability to efficiently ionize and image neutral lipids, such as triglycerides (TGs). To help overcome this obstacle, we have utilized silicon nanopost arrays (NAPA), a matrix-free laser desorption ionization (LDI) platform. Hidradenitis suppurativa (HS) is a chronic, recurrent inflammatory skin disease of the apocrine sweat glands. The ability of NAPA to efficiently ionize lipids is exploited in the analysis of human skin samples from sufferers of HS. Ionization by LDI from NAPA allows for the detection and imaging of a number of neutral lipid species, including TGs comprised of shorter, odd-chain fatty acids, which strongly suggests an increased bacterial load within the host tissue, as well as hexosylceramides (HexCers) and galabiosyl-/lactosylceramides that appear to be correlated with the presence of HS. Our results demonstrate that NAPA-LDI-MSI is capable of imaging and potentially differentiating healthy and diseased human skin tissues based on changes in detected neutral lipid composition.

Transitional cell carcinoma of the urethra [correction of ureter] in a patient with buttock pain: a case report.

This case reports on a patient with an unusual presentation of a rare tumor: urethral transitional cell carcinoma (TCC). Urethral TCC occurs in approximately 0.7% to 4.0% of patients who have had primary bladder cancer. The initial symptoms usually involve hematuria, with approximately a third of patients reporting flank area pain. Buttock pain and the absence of hematuria are uncommon with this disorder. The patient was initially suspected to have piriformis syndrome, but when he did not respond as expected to treatment, and because of his history of primary bladder cancer, further evaluation was undertaken and the diagnosis was made. The patient responded well to radiation and chemotherapy. Musculoskeletal physicians should be particularly suspicious of the presence of urethral TCC in a patient with a history of primary bladder cancer who reports low back or buttock pain, particularly if the patient does not respond quickly to treatment.

Syntaxin 6 regulates Glut4 trafficking in 3T3-L1 adipocytes.

Insulin stimulates the movement of glucose transporter-4 (Glut4)-containing vesicles to the plasma membrane of adipose cells. We investigated the role of post-Golgi t-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in the trafficking of Glut4 in 3T3-L1 adipocytes. Greater than 85% of syntaxin 6 was found in Glut4-containing vesicles, and this t-SNARE exhibited insulin-stimulated movement to the plasma membrane. In contrast, the colocalization of Glut4 with syntaxin 7, 8, or 12/13 was limited and these molecules did not translocate to the plasma membrane. We used adenovirus to overexpress the cytosolic domain of these syntaxin's and studied their effects on Glut4 traffic. Overexpression of the cytosolic domain of syntaxin 6 did not affect insulin-stimulated glucose transport, but increased basal deGlc transport and cell surface Glut4 levels. Moreover, the syntaxin 6 cytosolic domain significantly reduced the rate of Glut4 reinternalization after insulin withdrawal and perturbed subendosomal Glut4 sorting; the corresponding domains of syntaxins 8 and 12 were without effect. Our data suggest that syntaxin 6 is involved in a membrane-trafficking step that sequesters Glut4 away from traffic destined for the plasma membrane. We speculate that this is at the level of traffic of Glut4 into its unique storage compartment and that syntaxin 16 may be involved.

Cervical epidural abscess in an afebrile patient: a case report.

OBJECTIVE: The aim of the study was to discuss a case of cervical epidural abscess and present the clinical features and diagnosis of this disorder. CLINICAL FEATURES: A 52-year-old man had severe neck pain for 1 week. He had a long history of chronic recurrent neck pain, including 2 previous neck surgeries. He was afebrile on each visit, but developed rapid onset of motor loss, which necessitated immediate magnetic resonance imaging referral. The patient died in the magnetic resonance imaging tube. INTERVENTION AND OUTCOME: Because of the aggressive nature of the infection, causing death only 1 week after the initial onset of symptoms, there was no opportunity to institute treatment of the abscess. CONCLUSION: Spinal epidural abscess is a relatively rare but potentially life-threatening disorder in which the most common initial symptom is spinal pain. Because of this, patients with early spinal epidural abscess will often consult a chiropractic physician or other primary contact spine specialist. It is important for clinicians to be alert to the diagnostic features of spinal epidural abscess so that early treatment can be instituted.

Genome-wide ChIP-seq analysis of human TOP2B occupancy in MCF7 breast cancer epithelial cells.

We report the whole genome ChIP seq for human TOP2B from MCF7 cells. Using three different peak calling methods, regions of binding were identified in the presence or absence of the nuclear hormone estradiol, as TOP2B has been reported to play a role in ligand-induced transcription. TOP2B peaks were found across the whole genome, 50% of the peaks fell either within a gene or within 5 kb of a transcription start site. TOP2B peaks coincident with gene promoters were less frequently associated with epigenetic features marking active promoters in estradiol treated than in untreated cells. Significantly enriched transcription factor motifs within the DNA sequences underlying the peaks were identified. These included SP1, KLF4, TFAP2A, MYF, REST, CTCF, ESR1 and ESR2. Gene ontology analysis of genes associated with TOP2B peaks found neuronal development terms including axonogenesis and axon guidance were significantly enriched. In the absence of functional TOP2B there are errors in axon guidance in the zebrafish eye. Specific heparin sulphate structures are involved in retinal axon targeting. The glycosaminoglycan biosynthesis-heparin sulphate/heparin pathway is significantly enriched in the TOP2B gene ontology analysis, suggesting changes in this pathway in the absence of TOP2B may cause the axon guidance faults.

Adiponectin corrects high-fat diet-induced disturbances in muscle metabolomic profile and whole-body glucose homeostasis.

We provide here a detailed and comprehensive analysis of skeletal muscle metabolomic profiles in response to adiponectin in adiponectin knockout (AdKO) mice after high-fat-diet (HFD) feeding. Hyperinsulinemic-euglycemic clamp studies showed that adiponectin administration corrected HFD-induced defects in post/basal insulin stimulated R(d) and insulin signaling in skeletal muscle. Lipidomic profiling of skeletal muscle from HFD-fed mice indicated elevated triacylglycerol and diacylglycerol species (16:0-18:1, 18:1, and 18:0-18:2) as well as acetyl coA, all of which were mitigated by adiponectin. HFD induced elevated levels of various ceramides, but these were not significantly altered by adiponectin. Adiponectin corrected the altered branched-chain amino acid metabolism caused by HFD and corrected increases across a range of glycerolipids, fatty acids, and various lysolipids. Adiponectin also reversed induction of the pentose phosphate pathway by HFD. Analysis of muscle mitochondrial structure indicated that adiponectin treatment corrected HFD-induced pathological changes. In summary, we show an unbiased comprehensive metabolomic profile of skeletal muscle from AdKO mice subjected to HFD with or without adiponectin and relate these to changes in whole-body glucose handling, insulin signaling, and mitochondrial structure and function. Our data revealed a key signature of relatively normalized muscle metabolism across multiple metabolic pathways with adiponectin supplementation under the HFD condition.

MRE11 facilitates the removal of human topoisomerase II complexes from genomic DNA.

Topoisomerase II creates a double-strand break intermediate with topoisomerase covalently coupled to the DNA via a 5'-phosphotyrosyl bond. These intermediate complexes can become cytotoxic protein-DNA adducts and DSB repair at these lesions requires removal of topoisomerase II. To analyse removal of topoisomerase II from genomic DNA we adapted the trapped in agarose DNA immunostaining assay. Recombinant MRE11 from 2 sources removed topoisomerase IIα from genomic DNA in vitro, as did MRE11 immunoprecipitates isolated from A-TLD or K562 cells. Basal topoisomerase II complex levels were very high in A-TLD cells lacking full-length wild type MRE11, suggesting that MRE11 facilitates the processing of topoisomerase complexes that arise as part of normal cellular metabolism. In K562 cells inhibition of MRE11, PARP or replication increased topoisomerase IIα and ß complex levels formed in the absence of an anti-topoisomerase II drug.

Changes in protein expression profiles between a low phytic acid rice ( Oryza sativa L. Ssp. japonica) line and its parental line: a proteomic and bioinformatic approach.

The seed proteome of a low phytic acid (lpa) rice line (Os-lpa-XS110-1), developed as a novel food source, was compared to that of its parental line, Xiushui 110 (XS-110). Analysis by surfaced enhanced laser desorption ionization-time-of-flight mass spectrometry (SELDI-TOF MS) and two-dimensional gel electrophoresis (2-DE) allowed the detection of a potential low molecular weight biomarker and identification of 23 differentially expressed proteins that include stress-related proteins, storage proteins, and potential allergens. Bioinformatic analyses revealed that triose phosphate isomerase (TPI) and fructose bisphosphatealdolase (FBA), two major differentially expressed proteins, are involved in myo-inositol metabolism. Accumulation of globulin was also significantly decreased in the lpa line. This study demonstrates the potential of proteomic and bioinformatic profiling techniques for safety assessment of novel foods. Furthermore, these techniques provide powerful tools for studying functional genomics due to the possibility of identifying genes related to the mutated traits.