Gel-assisted mass spectrometry imaging enables sub-micrometer spatial lipidomics.

A technique capable of label-free detection, mass spectrometry imaging (MSI) is a powerful tool for spatial investigation of native biomolecules in intact specimens. However, MSI has often been precluded from single-cell applications due to the spatial resolution limit set forth by the physical and instrumental constraints of the method. By taking advantage of the reversible interaction between the analytes and a superabsorbent hydrogel, we have developed a sample preparation and imaging workflow named Gel-Assisted Mass Spectrometry Imaging (GAMSI) to overcome the spatial resolution limits of modern mass spectrometers. With GAMSI, we show that the spatial resolution of MALDI-MSI can be enhanced ~3-6-fold to the sub-micrometer level without changing the existing mass spectrometry hardware or analysis pipeline. This approach will vastly enhance the accessibility of MSI-based spatial analysis at the cellular scale.

Rewiring of cortical glucose metabolism fuels human brain cancer growth.

The brain avidly consumes glucose to fuel neurophysiology. Cancers of the brain, such as glioblastoma (GBM), lose aspects of normal biology and gain the ability to proliferate and invade healthy tissue. How brain cancers rewire glucose utilization to fuel these processes is poorly understood. Here we perform infusions of 13 C-labeled glucose into patients and mice with brain cancer to define the metabolic fates of glucose-derived carbon in tumor and cortex. By combining these measurements with quantitative metabolic flux analysis, we find that human cortex funnels glucose-derived carbons towards physiologic processes including TCA cycle oxidation and neurotransmitter synthesis. In contrast, brain cancers downregulate these physiologic processes, scavenge alternative carbon sources from the environment, and instead use glucose-derived carbons to produce molecules needed for proliferation and invasion. Targeting this metabolic rewiring in mice through dietary modulation selectively alters GBM metabolism and slows tumor growth. Significance: This study is the first to directly measure biosynthetic flux in both glioma and cortical tissue in human brain cancer patients. Brain tumors rewire glucose carbon utilization away from oxidation and neurotransmitter production towards biosynthesis to fuel growth. Blocking these metabolic adaptations with dietary interventions slows brain cancer growth with minimal effects on cortical metabolism.

Fully implanted battery-free high power platform for chronic spinal and muscular functional electrical stimulation.

Electrical stimulation of the neuromuscular system holds promise for both scientific and therapeutic biomedical applications. Supplying and maintaining the power necessary to drive stimulation chronically is a fundamental challenge in these applications, especially when high voltages or currents are required. Wireless systems, in which energy is supplied through near field power transfer, could eliminate complications caused by battery packs or external connections, but currently do not provide the harvested power and voltages required for applications such as muscle stimulation. Here, we introduce a passive resonator optimized power transfer design that overcomes these limitations, enabling voltage compliances of ± 20 V and power over 300 mW at device volumes of 0.2 cm2, thereby improving power transfer 500% over previous systems. We show that this improved performance enables multichannel, biphasic, current-controlled operation at clinically relevant voltage and current ranges with digital control and telemetry in freely behaving animals. Preliminary chronic results indicate that implanted devices remain operational over 6 weeks in both intact and spinal cord injured rats and are capable of producing fine control of spinal and muscle stimulation.

FluoMALDI Microscopy: Matrix Co-Crystallization Simultaneously Enhances Fluorescence and MALDI Imaging.

Here, the authors report that co-crystallization of fluorophores with matrix-assisted laser desorption/ionization (MALDI) imaging matrices significantly enhances fluorophore brightness up to 79-fold, enabling the amplification of innate tissue autofluorescence. This discovery facilitates FluoMALDI, the imaging of the same biological sample by both fluorescence microscopy and MALDI imaging. The approach combines the high spatial resolution and specific labeling capabilities of fluorescence microscopy with the inherently multiplexed, versatile imaging capabilities of MALDI imaging. This new paradigm simplifies registration by avoiding physical changes between fluorescence and MALDI imaging, allowing to image the exact same cells in tissues with both modalities. Matrix-fluorophore co-crystallization also facilitates applications with insufficient fluorescence brightness. The authors demonstrate  feasibility of FluoMALDI imaging with endogenous and exogenous fluorophores and autofluorescence-based FluoMALDI of brain and kidney tissue sections. FluoMALDI will advance structural-functional microscopic imaging in cell biology, biomedicine, and pathology.

RaMALDI: Enabling simultaneous Raman and MALDI imaging of the same tissue section.

Multimodal tissue imaging techniques that integrate two complementary modalities are powerful discovery tools for unraveling biological processes and identifying biomarkers of disease. Combining Raman spectroscopic imaging (RSI) and matrix-assisted laser-desorption/ionization (MALDI) mass spectrometry imaging (MSI) to obtain fused images with the advantages of both modalities has the potential of providing spatially resolved, sensitive, specific biomolecular information, but has so far involved two separate sample preparations, or even consecutive tissue sections for RSI and MALDI MSI, resulting in images with inherent disparities. We have developed RaMALDI, a streamlined, integrated, multimodal imaging workflow of RSI and MALDI MSI, performed on a single tissue section with one sample preparation protocol. We show that RaMALDI imaging of various tissues effectively integrates molecular information acquired from both RSI and MALDI MSI of the same sample, which will drive discoveries in cell biology, biomedicine, and pathology, and advance tissue diagnostics.

Rapid and stain-free quantification of viral plaque via lens-free holography and deep learning.

A plaque assay-the gold-standard method for measuring the concentration of replication-competent lytic virions-requires staining and usually more than 48 h of runtime. Here we show that lens-free holographic imaging and deep learning can be combined to expedite and automate the assay. The compact imaging device captures phase information label-free at a rate of approximately 0.32 gigapixels per hour per well, covers an area of about 30 × 30 mm2 and a 10-fold larger dynamic range of virus concentration than standard assays, and quantifies the infected area and the number of plaque-forming units. For the vesicular stomatitis virus, the automated plaque assay detected the first cell-lysing events caused by viral replication as early as 5 h after incubation, and in less than 20 h it detected plaque-forming units at rates higher than 90% at 100% specificity. Furthermore, it reduced the incubation time of the herpes simplex virus type 1 by about 48 h and that of the encephalomyocarditis virus by about 20 h. The stain-free assay should be amenable for use in virology research, vaccine development and clinical diagnosis.

Co-mutation of ASXL1 and SF3B1 Predicts Poorer Overall Survival Than Isolated ASXL1 or SF3B1 Mutations.

BACKGROUND/AIM: Mutations in the ASXL transcriptional regulator 1 (ASXL1) and splicing factor 3b subunit 1(SF3B1) genes are commonly observed in myeloid neoplasms and are independent predicative factors for overall survival (OS). Only a few contradictory reports exist on the clinical significance of concurrent ASXL1 and SF3B1 mutations. Previous studies also did not exclude patients with mutations of other genes, which could be confounding factors. MATERIALS AND METHODS: We identified 69 patients with mutation of only ASXL1, 89 patients with mutation of only SF3B1, and 17 patients with mutations exclusively of both ASXL1 and SF3B1 from our database of 8,285 patients and compared their clinical features and outcomes. RESULTS: Patients with ASXL1 mutations more frequently had acute myeloid leukemia (22.47%) or clonal cytopenia of unknown significance than patients with SF3B1 mutations (1.45%) or with ASXL1/SF3B1 mutations (11.76%). Patients with SF3B1 or ASXL1/SF3B1 mutations were more frequently diagnosed with myelodysplastic syndrome (75.36% and 64.71%, respectively) than patients with ASXL1 mutations (24.72%). Patients with ASXL1/SF3B1 (23.53%) mutations more frequently had myelodysplastic/myeloid proliferative neoplasm than did patients with ASXL1 mutations (5.62%) or with SF3B1 mutations (15.94%). OS of the ASXL1 mutation-only group was worse than that of the SF3B1 mutation-only group with a hazard ratio of 5.83 (p=0.017). Finally, and most importantly, the OS of the ASXL1/SF3B1 co-mutation group was poorer than that of both single-mutation groups (p=0.005). CONCLUSION: ASXL1/SF3B1 co-mutations portend worse OS than isolated ASXL1 or SF3B1 mutations, which might be due to abnormalities in both the epigenetic-regulatory and RNA-splicing pathways or because two genes instead of one are mutated.

A multimodal pipeline using NMR spectroscopy and MALDI-TOF mass spectrometry imaging from the same tissue sample.

NMR spectroscopy and matrix assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) are both commonly used to detect large numbers of metabolites and lipids in metabolomic and lipidomic studies. We have demonstrated a new workflow, highlighting the benefits of both techniques to obtain metabolomic and lipidomic data, which has realized for the first time the combination of these two complementary and powerful technologies. NMR spectroscopy is frequently used to obtain quantitative metabolite information from cells and tissues. Lipid detection is also possible with NMR spectroscopy, with changes being visible across entire classes of molecules. Meanwhile, MALDI MSI provides relative measures of metabolite and lipid concentrations, mapping spatial information of many specific metabolite and lipid molecules across cells or tissues. We have used these two complementary techniques in combination to obtain metabolomic and lipidomic measurements from triple-negative human breast cancer cells and tumor xenograft models. We have emphasized critical experimental procedures that ensured the success of achieving NMR spectroscopy and MALDI MSI in a combined workflow from the same sample. Our data show that several phospholipid metabolite species were differentially distributed in viable and necrotic regions of breast tumor xenografts. This study emphasizes the power of combined NMR spectroscopy-MALDI imaging to advance metabolomic and lipidomic studies.

The cholesteryl ester transfer protein (CETP) raises cholesterol levels in the brain.

The cholesteryl ester transfer protein (CETP) is a lipid transfer protein responsible for the exchange of cholesteryl esters and triglycerides between lipoproteins. Decreased CETP activity is associated with longevity, cardiovascular health, and maintenance of good cognitive performance. Interestingly, mice lack the CETP-encoding gene and have very low levels of LDL particles compared with humans. Currently, the molecular mechanisms induced because of CETP activity are not clear. To understand how CETP activity affects the brain, we utilized CETP transgenic (CETPtg) mice that show elevated LDL levels upon induction of CETP expression through a high-cholesterol diet. CETPtg mice on a high-cholesterol diet showed up to 22% higher cholesterol levels in the brain. Using a microarray on mostly astrocyte-derived mRNA, we found that this cholesterol increase is likely not because of elevated de novo synthesis of cholesterol. However, cholesterol efflux is decreased in CETPtg mice along with an upregulation of the complement factor C1Q, which plays a role in neuronal cholesterol clearance. Our data suggest that CETP activity affects brain health through modulating cholesterol distribution and clearance. Therefore, we propose that CETPtg mice constitute a valuable research tool to investigate the impact of cholesterol metabolism on brain function.

CEST MRI and MALDI imaging reveal metabolic alterations in the cervical lymph nodes of EAE mice.

BACKGROUND: Multiple sclerosis (MS) is a neurodegenerative disease, wherein aberrant immune cells target myelin-ensheathed nerves. Conventional magnetic resonance imaging (MRI) can be performed to monitor damage to the central nervous system that results from previous inflammation; however, these imaging biomarkers are not necessarily indicative of active, progressive stages of the disease. The immune cells responsible for MS are first activated and sensitized to myelin in lymph nodes (LNs). Here, we present a new strategy for monitoring active disease activity in MS, chemical exchange saturation transfer (CEST) MRI of LNs. METHODS AND RESULTS: We studied the potential utility of conventional (T2-weighted) and CEST MRI to monitor changes in these LNs during disease progression in an experimental autoimmune encephalomyelitis (EAE) model. We found CEST signal changes corresponded temporally with disease activity. CEST signals at the 3.2 ppm frequency during the active stage of EAE correlated significantly with the cellular (flow cytometry) and metabolic (mass spectrometry imaging) composition of the LNs, as well as immune cell infiltration into brain and spinal cord tissue. Correlating primary metabolites as identified by matrix-assisted laser desorption/ionization (MALDI) imaging included alanine, lactate, leucine, malate, and phenylalanine. CONCLUSIONS: Taken together, we demonstrate the utility of CEST MRI signal changes in superficial cervical LNs as a complementary imaging biomarker for monitoring disease activity in MS. CEST MRI biomarkers corresponded to disease activity, correlated with immune activation (surface markers, antigen-stimulated proliferation), and correlated with LN metabolite levels.

Unlabeled aspirin as an activatable theranostic MRI agent for breast cancer.

Rationale: Chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) is emerging as an alternative to gadolinium-based contrast MRI. We have evaluated the possibility of CEST MRI of orthotopic breast tumor xenografts with unlabeled aspirin's conversion to salicylic acid (SA) through various enzymatic activities, most notably inhibition of cyclooxygenase (COX)-1/-2 enzymes. Methods: We measured the COX-1/-2 expression in four breast cancer cell lines by Western Blot analysis and selected the highest and lowest expressing cell lines. We then performed CEST MRI following aspirin treatment to detect SA levels and ELISA to measure levels of downstream prostaglandin E2 (PGE2). We also injected aspirin into the tail vein of mice growing orthotopic tumor xenografts which expressed high and low COX-1/-2 and acquired SA CEST MR images of these tumor xenografts for up to 70 minutes. Tumors were then harvested to perform Western Blot and ELISA experiments to measure COX-1/-2 expression and PGE2 levels, respectively. Results: Western Blots determined that SUM159 cells contained significantly higher COX-1/-2 expression levels than MDA-MB-231 cells, in line with higher levels of downstream PGE2. SA CEST MRI yielded similar contrast at approximately 3% for both cell lines, independent of COX-1/-2 expression level. PGE2 levels decreased by about 50% following aspirin treatment. Results from our mouse study aligned with cultured cells, the overall SA CEST MRI contrast in both MDA-MB-231 and SUM159 tumor xenograft models was 5~8% at one hour post injection. PGE2 levels were ten times higher in SUM159 than MDA-MB-231 and decreased by 50%. The CEST contrast directly depended on the injected dose, with ~6%, ~3% and ~1.5% contrast observed following injection of 100 µL of 300 mM, 200 mM and 150 mM aspirin, respectively. Conclusions: Our data demonstrate the feasibility of using aspirin as a noninvasive activatable CEST MRI contrast agent for breast tumor detection.

Factorial Design to Optimize Matrix Spraying Parameters for MALDI Mass Spectrometry Imaging.

Matrix deposition is a critical step in obtaining reproducible and spatially representative matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging data. To date, few limited studies have examined the optimization of matrix spraying parameters for maximizing analyte extraction while minimizing analyte delocalization. Herein, we present a study using automated pneumatic spraying with a heated sample-holder tray to determine an optimized model for mouse whole kidney lipid imaging using a 2,5-dihydroxybenzoic acid matrix in which the solvent flow rate, nozzle velocity, and sample heating were optimized using a two-level factorial experimental design. Parameters examined to determine the optimum model include the number of analytes, the matrix crystal size, off tissue delocalization, the signal intensity, and spray time. Our results show that sample heating using a heated tray while spraying improves the MALDI imaging performance. This improvement is possible because higher solvent flow rates can be used in the pneumatic sprayer, allowing for better sample extraction, while sample delocalization is minimized due to sample heating.

Visualizing the distribution of strawberry plant metabolites at different maturity stages by MALDI-TOF imaging mass spectrometry.

This study aimed to visualize differences in the distribution of citric acid, soluble sugars, and anthocyanins in strawberries at four different maturity stages (green to red strawberries) by matrix-assisted laser desorption/ionization time-of-flight imaging mass spectrometry (MALDI-TOF IMS). Results demonstrated citric acid and sugars are evenly distributed in the entire fruit at all maturity stages, while most of anthocyanins are mainly located in the periphery of fruit with increased abundance in red strawberries, indicating a correlation with the colour attributes. Sugar in red strawberries (11.92 brix) increased by two-fold compared to the green ones (6.23 brix). Finally, absolute quantitation of each compound from HPLC analyses support the quantitative results from MALDI-TOF IMS. The results provide a deeper understanding in the changes and distribution of phytochemicals during the growth of strawberries, and demonstrates the usefulness of IMS for plant breeding and postharvest technology.

C-C Motif Chemokine 8 promotes angiogenesis in vascular endothelial cells.

OBJECTIVES: Angiogenesis is an important progress associated with several pathological situations. Several chemokines have been reported to act as regulators of angiogenesis. The current study aimed to find whether C-C Motif Chemokine 8 is involved in angiogenesis regulation. METHODS: To verify whether C-C Motif Chemokine 8 is related to angiogenesis in plaques, carotid plaques were collected from patients with severe carotid stenosis and analysed using CD31 immunohistochemistry and real-time PCR. To further clarify the relation between C-C Motif Chemokine 8 and angiogenesis, human umbilical vein endothelium cells and human dermal microvascular endothelial cells were treated with C-C Motif Chemokine 8 in the presence or absence of C-C motif chemokine receptor 2-Ab and extracellular regulated MAP kinase 1/2 inhibition (FR180204). Proliferation and migration of human umbilical vein endothelium cells and human dermal microvascular endothelial cells were examined with Cell Counting Kit-8 and Transwell chamber assay, respectively. In vitro angiogenesis stimulated by C-C Motif Chemokine 8 was examined using tube formation assay. Ex vivo and in vivo angiogenesis were assessed by mice aortic ring assay and Matrigel plug assay, respectively. C-C motif chemokine receptors of human umbilical vein endothelium cells were examined with real-time PCR, and C-C motif chemokine receptor 1, C-C motif chemokine receptor 2, extracellular regulated MAP kinase 1/2 and phosphorylation-extracellular regulated MAP kinase 1/2 were examined with western blotting assay. RESULTS: C-C Motif Chemokine 8 was increased in carotid plaques with severe angiogenesis in both RNA and protein level. C-C Motif Chemokine 8 (5 ng/ml) weakly increased human umbilical vein endothelium cell proliferation, but not on human dermal microvascular endothelial cells. Migration and tube formation could be induced by C-C Motif Chemokine 8 in both human umbilical vein endothelium cells and human dermal microvascular endothelial cells. In mice aortic ring assay and Matrigel plug assay, C-C Motif Chemokine 8 could promote angiogenesis compared to vehicle groups. Phosphorylation of extracellular regulated MAP kinase 1/2 was increased with C-C Motif Chemokine 8 stimulation. The migration and tube formation promoted by C-C Motif Chemokine 8 could be largely blocked by C-C motif chemokine receptor 2-Ab or extracellular regulated MAP kinase 1/2 inhibition (FR180204). CONCLUSIONS: C-C Motif Chemokine 8 could promote both in vitro and in vivo angiogenesis. C-C motif chemokine receptor 2 played an important role in the activation of C-C Motif Chemokine 8 and extracellular regulated MAP kinase 1/2 signalling pathway was involved in this mechanism.

Contemporary Outcomes of Open and Endovascular Intervention for Extracranial Carotid Artery Aneurysms: A Single Centre Experience.

OBJECTIVE: The objective of this study was to evaluate the outcomes of open surgery (OS) and endovascular surgery (ES) for extracranial carotid aneurysm (ECCA) in the authors' centre. METHODS: Fifty-seven consecutive patients who were diagnosed with ECCA and underwent intervention from January 2005 to July 2019 at Zhongshan Hospital, Fudan University, were reviewed retrospectively. Patient characteristics and surgical outcomes for OS and ES were analysed. ECCAs were divided into three morphological subgroups: subgroup Ⅰ, no severe tortuosity of the internal carotid artery (ICA) or common carotid artery (CCA) proximal to the aneurysm, tortuosity of the aneurysm and 1 cm of peri-aneurysmal carotid artery ≤ 90°; subgroup Ⅱ, severe ICA or CCA tortuosity proximal to the aneurysm, tortuosity of the aneurysm and 1 cm of peri-aneurysmal carotid artery ≤ 90°; subgroup Ⅲ, aneurysm tortuosity and 1 cm peri-aneurysmal carotid artery > 90°. RESULTS: 35 patients underwent OS, 20 patients underwent ES and 2 patients underwent OS after the failure of ES. Thirty-six cases were classified in subgroup Ⅰ, 11 cases in subgroup Ⅱ, and 10 cases in subgroup Ⅲ. ES was achieved successfully in all 18 cases of subgroup I, but failed in three of four cases in subgroups Ⅱ and Ⅲ. With a mean duration of 62.9 ± 44.5 months of follow up, five deaths were recorded in the OS group, two of which were caused by ipsilateral stroke and three were not neurologically related. There was no stroke or death in the ES group during follow up. One case of stroke and two cases of death occurred in symptomatic patients, while one case of stroke and three cases of death occurred in asymptomatic patients. CONCLUSION: This series demonstrates that ES may be a safe and durable option for ECCA in subgroup Ⅰ, while in subgroups Ⅱ and Ⅲ, ES alone may be difficult to apply. A 30 day stroke rate around 5% existed in ECCAs with interventions, which should be considered before the intervention.

In vitro platform establishes antigen-specific CD8+ T cell cytotoxicity to encapsulated cells via indirect antigen recognition.

The curative potential of non-autologous cellular therapy is hindered by the requirement of anti-rejection therapy. Cellular encapsulation within nondegradable biomaterials has the potential to inhibit immune rejection, but the efficacy of this approach in robust preclinical and clinical models remains poor. While the responses of innate immune cells to the encapsulating material have been characterized, little attention has been paid to the contributions of adaptive immunity in encapsulated graft destabilization. Avoiding the limitations of animal models, we established an efficient, antigen-specific in vitro platform capable of delineating direct and indirect host T cell recognition to microencapsulated cellular grafts and evaluated their consequential impacts. Using ovalbumin (OVA) as a model antigen, we determined that alginate microencapsulation abrogates direct CD8+ T cell activation by interrupting donor-host interaction; however, indirect T cell activation, mediated by host antigen presenting cells (APCs) primed with shed donor antigens, still occurs. These activated T cells imparted cytotoxicity on the encapsulated cells, likely via diffusion of cytotoxic solutes. Overall, this platform delivers unique mechanistic insight into the impacts of hydrogel encapsulation on host adaptive immune responses, comprehensively addressing a long-standing hypothesis of the field. Furthermore, it provides an efficient benchtop screening tool for the investigation of new encapsulation methods and/or synergistic immunomodulatory agents.

Toward the Rational Design of Universal Dual Polarity Matrix for MALDI Mass Spectrometry.

A series of novel anthranilic acid derivatives I-IV, of which COOH-NH2 (I) and COOH-NHMe (IV) are endowed with acid and base bifunctionality, were designed and synthesized for matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry applications in dual polarity molecular imaging of biological samples, particularly for lipids. The heat of protonation, deprotonation, and proton transfer reaction as well as the capability of analyzing biomolecules in both positive and negative ion modes for I-IV were systematically investigated under standard 355 nm laser excitation. The results indicate correlation between dual polarity and acid-base property. Further, COOH-NHMe (IV) showed a unique performance and was successfully applied as the matrix for MALDI-TOF mass spectrometry imaging (MSI) for studying the mouse brain. Our results demonstrate the superiority of COOH-NHMe (IV) in detecting more lipid and protein species compared to commercially available matrices. Moreover, MALDI-TOF MSI results were obtained for lipid distributions, making COOH-NHMe (IV) a potential next generation universal matrix.

Minimizing Visceral Fat Delocalization on Tissue Sections with Porous Aluminum Oxide Slides for Imaging Mass Spectrometry.

A high correlation of bioanalytes with their corresponding histologies is the landmark feature of matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS). Lipids are one of the most studied classes of biomolecules, and monitoring lipid distribution and abundance in tissue samples can lead to major inputs in the understanding of disease. Lipid delocalization and ion suppression are two major effects that can lead to misinterpretation of the IMS results to an unaware analyst. We and others have observed that tissue specimens containing high amounts of visceral fat are challenging to analyze because of fat delocalization on and off section leading to significant triacylglyceride and phospholipid delocalization and major ion suppression effects. In this work, we introduce a novel and easy to produce reusable porous aluminum oxide sample slide that minimizes visceral fat delocalization after thaw-mounting of tissue sections. Using fatty mouse kidneys and other tissues, we demonstrate its efficacy in minimizing delocalization of triacylglycerides, the primary constituents of fat, and the resulting beneficial effects on phospholipid MALDI IMS.

Silver spray deposition for AgLDI imaging MS of cholesterol and other olefins on thin tissue sections.

Olefins such as cholesterol and unsaturated fatty acids play important biological roles. Silver-assisted laser desorption ionization (AgLDI) takes advantage of the strong affinity of silver to conjugate with double bonds to selectively ionize these molecules for imaging mass spectrometry (IMS) experiments. For IMS studies, two main approaches for silver deposition have been described in the literature: fine coating by silver sputtering and spray deposition of silver nanoparticles. While these approaches allow for extremely high resolution IMS experiments to be conducted, they are not readily available to all laboratories. Herein, we present a silver nitrate spray deposition approach as an alternative to silver sputtering and nanoparticle deposition for routine IMS analysis. The silver nitrate spray has the same level of specificity and sensitivity for olefins, particularly cholesterol, and has shown to be capable of IMS experiments down to 10-µm spatial resolution. Minimal sample preparation and the affordability of silver nitrate make this a convenient and accessible technique worth considering.

A Systematic Review and Updated Metaanalysis for Carotid Near-Occlusion.

BACKGROUND: Carotid near-occlusion (CNO) is distal luminal collapse of the internal carotid artery beyond a tight stenosis. CNO is a relatively rare condition accounting for 3% in symptomatic carotid stenosis and about 20% in severe (≥70%) symptomatic stenosis. The optimal treatment for CNO remains controversial. METHODS: This systematic review and metaanalysis were performed in accordance with the Meta-analysis of Observational Studies in Epidemiology guidelines (MOOSE). We searched MEDLINE, the Cochrane Library, and EMBASE for articles published from inception date to November 2018. Methodological Index for Non-randomized Studies (MINORS) was used to evaluate the methodological quality of studies. We defined primary outcome as any stroke, death and myocardial infarction (MI) within 30 days after intervention and the operative risks of carotid endarterectomy (CEA) and carotid artery stenting (CAS) were evaluated by the incidence rate (IR) of the primary outcome. Secondary outcome was defined as ipsilateral stroke, neurogenic or cardiac death and MI during the follow-up. Long-term risk was evaluated by the IR of secondary outcome. The analyses used the IRs of secondary outcome and restenosis per person-year (p-y) were performed to evaluate long-term risk and restenosis. Pooled analyses of different therapy groups were calculated. RESULTS: Twenty-eight articles of 26 studies met the inclusion criteria and were eligible for pooled analysis. Pooled IR of secondary outcome was 4.26 per 100 p-ys (95% CI, 2.92-6.20 per 100 p-ys) in intervention group (heterogeneity, I2 = 56.1%, P < 0.01; Egger test, P = 0.73) and 13.3 per 100 p-ys (95% CI, 5.54-31.95 per 100 p-ys) in best medical treatment (BMT) group (heterogeneity, I2 = 88.3%, P < 0.01; Egger test, P = 0.76). No significant difference was demonstrated in operative risk (CEA: 4.82%, 95% confidence interval [CI]: 3.07-7.55%; CAS: 5.39%, 95% CI: 3.69-7.88%) and long-term risk (CEA: 4.47 per 100 p-ys, 95% CI: 3.35-5.97 per 100 p-ys; CAS: 4.71 per 100 p-ys, 95% CI: 2.37-9.37 per 100 p-ys) between CEA and CAS group. CONCLUSIONS: BMT alone may be not enough to support a better prognosis than CEA or CAS for patients with CNO. No significant difference was found between patients with CNO who underwent CAS and CEA in both perioperative period and long-term follow-up.