Next-generation pathology practices with mass spectrometry imaging.

Mass spectrometry imaging (MSI) is a powerful technique that reveals the spatial distribution of various molecules in biological samples, and it is widely used in pathology-related research. In this review, we summarize common MSI techniques, including matrix-assisted laser desorption/ionization and desorption electrospray ionization MSI, and their applications in pathological research, including disease diagnosis, microbiology, and drug discovery. We also describe the improvements of MSI, focusing on the accumulation of imaging data sets, expansion of chemical coverage, and identification of biological significant molecules, that have prompted the evolution of MSI to meet the requirements of pathology practices. Overall, this review details the applications and improvements of MSI techniques, demonstrating the potential of integrating MSI techniques into next-generation pathology practices.

Multiplexed electrical detection of whole viruses from plasma in a microfluidic platform.

The advancement of point-of-care diagnostics is crucial to improving patient outcomes, especially in areas with low access to hospitals or specialized laboratories. In particular, rapid, sensitive, and multiplexed detection of disease biomarkers has great potential to achieve accurate diagnosis and inform high quality care for patients. Our Coulter counting and immunocapture based detection system has previously shown its broad applicability in the detection of cells, proteins, and nucleic acids. This paper expands the capability of the platform by demonstrating multiplexed detection of whole-virus particles using electrically distinguishable hydrogel beads by demonstrating the capability of our platform to achieve simultaneous detection at clinically relevant concentrations of hepatitis A virus (>2 × 103 IU mL-1) and human parvovirus B19 virus like particles (>106 IU mL-1) from plasma samples. The expanded versatility of the differential electrical counting platform allows for more robust and diverse testing capabilities.

Predicting Breast Cancer by Paper Spray Ion Mobility Spectrometry Mass Spectrometry and Machine Learning.

Paper spray ionization has been used as a fast sampling/ionization method for the direct mass spectrometric analysis of biological samples at ambient conditions. Here, we demonstrated that by utilizing paper spray ionization-mass spectrometry (PSI-MS) coupled with field asymmetric waveform ion mobility spectrometry (FAIMS), predictive metabolic and lipidomic profiles of routine breast core needle biopsies could be obtained effectively. By the combination of machine learning algorithms and pathological examination reports, we developed a classification model, which has an overall accuracy of 87.5% for an instantaneous differentiation between cancerous and noncancerous breast tissues utilizing metabolic and lipidomic profiles. Our results suggested that paper spray ionization-ion mobility spectrometry-mass spectrometry (PSI-IMS-MS) is a powerful approach for rapid breast cancer diagnosis based on altered metabolic and lipidomic profiles.

Multimodal Imaging of Amyloid Plaques: Fusion of the Single-Probe Mass Spectrometry Image and Fluorescence Microscopy Image.

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases. The formation of amyloid plaques by aggregated amyloid beta (Aß) peptides is a primary event in AD pathology. Understanding the metabolomic features and related pathways is critical for studying plaque-related pathological events (e.g., cell death and neuron dysfunction). Mass spectrometry imaging (MSI), due to its high sensitivity and ability to obtain the spatial distribution of metabolites, has been applied to AD studies. However, limited studies of metabolites in amyloid plaques have been performed due to the drawbacks of the commonly used techniques such as matrix-assisted laser desorption/ionization MSI. In the current study, we obtained high spatial resolution (∼17 µm) MS images of the AD mouse brain using the Single-probe, a microscale sampling and ionization device, coupled to a mass spectrometer under ambient conditions. The adjacent slices were used to obtain fluorescence microscopy images to locate amyloid plaques. The MS image and the fluorescence microscopy image were fused to spatially correlate histological protein hallmarks with metabolomic features. The fused images produced significantly improved spatial resolution (∼5 µm), allowing for the determination of fine structures in MS images and metabolomic biomarkers representing amyloid plaques.

Intravenous Thrombolysis in Acute Ischemic Stroke After Idarucizumab Reversal of Dabigatran Effect: Analysis of the Cases From Taiwan.

BACKGROUND: Asians with atrial fibrillation carry a higher risk of ischemic stroke than non-Asians even under treatment of nonvitamin K antagonist oral anticoagulants. The purpose of the study was to observe the feasibility of intravenous thrombolytic therapy after administering a reversal agent, idarucizumab, in dabigatran-treated patients with acute ischemic stroke in Taiwan. METHODS: Dabigatran-treated patients with acute ischemic stroke who received intravenous recombinant tissue plasminogen activator (rt-PA) after idarucizumab reversal were enrolled in the retrospective nationwide study. The clinical data, treatment course, and outcomes were recorded. Stroke severity was evaluated using the National Institutes of Health Stroke Scale (NIHSS) score. Any intracerebral hemorrhage (ICH) after rt-PA was detected by neuroimaging studies. RESULTS: Ten dabigatran-treated patients (6 men, mean age 71.10 ± 7.96 years) with acute ischemic stroke were included. Before stroke, the mean CHA2DS2-VASc score was 4.50 ± 1.57 and 8 patients (80%) received dabigatran 110 mg twice daily. All patients were treated with 5 g idarucizumab, following which the activated partial thromboplastin time normalized. Intravenous rt-PA (mean dose .78 mg/kg) was initiated a mean time of 11.11 minutes after idarucizumab infusion. The NIHSS score improved significantly after thrombolysis (16.0 ± 6.67 at admission to 9.38 ± 4.75 at discharge, P = .016). ICH developed in 3 patients (30%). Two of them were asymptomatic and 1 patient suffered from symptomatic ICH leading to mortality. CONCLUSION: Our data reconfirmed the feasibility of intravenous rt-PA for Asian stroke patients after reversal of dabigatran effect with idarucizumab.

Facile multi-dimensional profiling of chemical gradients at the millimetre scale.

A vast number of conventional physicochemical methods are suitable for the analysis of homogeneous samples. However, in various cases, the samples exhibit intrinsic heterogeneity. Tomography allows one to record approximate distributions of chemical species in the three-dimensional space. Here we develop a simple optical tomography system which enables performing scans of non-homogeneous samples at different wavelengths. It takes advantage of inexpensive open-source electronics and simple algorithms. The analysed samples are illuminated by a miniature LCD/LED screen which emits light at three wavelengths (598, 547 and 455 nm, corresponding to the R, G, and B channels, respectively). On presentation of every wavelength, the sample vial is rotated by ∼180°, and videoed at 30 frames per s. The RGB values of pixels in the obtained digital snapshots are subsequently collated, and processed to produce sinograms. Following the inverse Radon transform, approximate quasi-three-dimensional images are reconstructed for each wavelength. Sample components with distinct visible light absorption spectra (myoglobin, methylene blue) can be resolved. The system was used to follow dynamic changes in non-homogeneous samples in real time, to visualize binary mixtures, to reconstruct reaction-diffusion fronts formed during the reduction of 2,6-dichlorophenolindophenol by ascorbic acid, and to visualize the distribution of fungal mycelium grown in a semi-solid medium.

Unbiased, Cell-free Profiling of Single Influenza Genomes at High-throughput.

The segmented structure of the Influenza A virus (IAV) genome facilitates reassortment, segment exchange during co-infection. When divergent strains mix across human, agricultural, and wildlife reservoirs novel strains are generated, which has been the source of pandemics. Due to the limited throughput and infection-based assays, IAV reassortment studies has been limited to permissive reassortment. We have developed DE-flowSVP to achieve extremely high throughput, direct profiling of as many as 10 5 IAV particles in a single-day experiment and enabled quantitative profiling of reassortment propensity between divergent strains for the first time. By profiling reassortants between two naturally circulating low-pathogenicity avian IAVs, we confirmed that molecular incompatibility yields strong preference toward within-strain mixing. Surprisingly, we revealed that two-to-three particle aggregation contributed primarily to genome mixing (75-99%), suggesting that aggregation mediated by sialic acid binding by viral surface proteins provides a secondary pathway to genome mixing while avoiding the co-packaging fitness cost. We showed that genome mixing is sensitively dependent on co-infection timing, relative segment abundances, and viral surface-protein background. DE-flowSVP enables large-scale survey of reassortment potential among the broad diversity of IAV strains informing pandemic strain emergence.

Real-time bottom-up characterization of protein mixtures enabled by online microdroplet-assisted enzymatic digestion (MAED).

Enzymatic digestion remains one of the "rate-determining steps" in the bottom-up analysis of proteins. However, by performing digestion in microdroplets generated from electrosonic spray, the reaction could be accelerated to a timescale lower than milliseconds. Here, we describe a simple and rapid online digestion platform named online microdroplet-assisted enzymatic digestion (MAED). It involves the integration of intact protein separation with enzymatic digestion in microdroplets. Via online MAED, various protein standards, including an antibody standard, were characterized in a bottom-up manner without prior digestion, and high sequence coverages were obtained. We further extended the application of online MAED to a more complex sample, mouse brain extract, where protein identifications were successfully yielded. Compared with the conventional bottom-up approach, a more comprehensive characterization could be obtained particularly for low molecular weight proteins. In short, we provide a rapid and alternative bottom-up analysis in a top-down fashion as well as a new possibility for microdroplet chemistry.

Electrochemical point-of-care devices for the diagnosis of sepsis.

Sepsis is a life-threatening dysfunction of organ systems caused by a dysregulated immune system because of an infectious process. It remains one of the leading causes of hospital mortality and of hospital readmissions in the United States. Mortality from sepsis increases with each hour of delayed treatment, therefore, diagnostic devices that can reduce the time from the onset of a patient's infection to the delivery of appropriate therapy are urgently needed. Likewise, tools that are capable of high-frequency testing of clinically relevant biomarkers are required to study disease progression. Electrochemical biosensors offer important advantages such as high sensitivity, fast response, miniaturization, and low cost that can be adapted to clinical needs. In this review paper, we discuss the current state, limitations, and future directions of electrochemical-based point-of-care detection platforms that contribute to the diagnosis and monitoring of sepsis.

Prevalence and Associated Factors of Visual Hallucinations in Patients with Vascular Cognitive Impairment.

Visual hallucinations (VHs) are striking features for dementia, especially dementia with Lewy bodies (DLB). We aimed to study the frequency and associated factors of VH in vascular cognitive impairment (VCI) and investigate the feasibility of clinically diagnosing the mixed pathology of VCI with DLB. This is a multicentre registration study. A consecutive series of VCI patients with and without dementia were enrolled. Frequency of VH and associated factors, including age, gender, education, disease severity, DLB clinical features, vascular risk factors, cognitive function, and neuropsychiatric symptoms, were compared between VCI with VH (VH+) and without VH (VH-). Among the 1281 patients analysed, 155 (12.1%) had VH. The VH+ group was older (t = 5.07; p < 0.001), was more likely to be female (χ 2 = 13.46; p < 0.001), and has a higher clinical dementia rating (χ 2 = 70.51; p < 0.001). After adjusting for age, gender, and disease severity, the VH+ group had poorer cognition and more severe neuropsychiatric symptoms. The VH+ group was more associated with DLB features in fluctuating cognition (OR = 2.48; p < 0.001), parkinsonism (OR = 1.85; p = 0.001), rapid eye movement (REM) behavioral disorder (OR = 4.56; p < 0.001), and ≧2 DLB core features (OR = 26.01; p < 0.001). VCI patients with VH tend to have more severe dementia, neuropsychiatric symptoms, and poorer cognitive function. Additionally, highly associated with clinical DLB features in VCI with VH raised the possibility of mixed pathology with DLB in this group. More than two core features in VCI might help in diagnosing a mixed pathology with DLB.

Remodeling nanoDESI Platform with Ion Mobility Spectrometry to Expand Protein Coverage in Cancerous Tissue.

Nanospray desorption electrospray ionization mass spectrometry is an ambient ionization technique that is capable of mapping proteins in tissue sections. However, high-abundant molecules or isobaric interference in biological samples hampers its broad applications in probing low-abundant proteins. To address this challenge, herein we demonstrated an integrated module that coupled pneumatic-assisted nanospray desorption electrospray ionization mass spectrometry with high-field asymmetric ion mobility spectrometry. Using this module to analyze mouse brain sections, the protein coverage was significantly increased. This improvement allowed the mapping of low-abundant proteins in tissue sections with a 5 µm spatial resolution enabled by computationally assisted fusion with optical microscopic images. Moreover, the module was successfully applied to characterize melanoma in skin tissues based on the enhanced protein profiles. The results suggested that this integrating module will be potentially applied to discover novel proteins in cancers.

Counteracting Cisplatin-Induced Testicular Damages by Natural Polyphenol Constituent Honokiol.

Cisplatin, despite its anti-cancer ability, exhibits severe testicular toxicities when applied systemically. Due to its wide application in cancer treatment, reduction of its damages to normal tissue is an imminent clinical need. Here we evaluated the effects of honokiol, a natural lipophilic polyphenol compound, on cisplatin-induced testicular injury. We showed in-vitro and in-vivo that nanosome-encapsulated honokiol attenuated cisplatin-induced DNA oxidative stress by suppressing intracellular reactive oxygen species production and elevating gene expressions of mitochondrial antioxidation enzymes. Nanosome honokiol also mitigated endoplasmic reticulum stress through down regulation of Bip-ATF4-CHOP signaling pathway. Additionally, this natural polyphenol compound diminished cisplatin-induced DNA breaks and cellular apoptosis. The reduced type I collagen accumulation in the testis likely attributed from inhibition of TGFß1, αSMA and ER protein TXNDC5 protein expression. The combinatorial beneficial effects better preserve spermatogenic layers and facilitate repopulation of sperm cells. Our study renders opportunity for re-introducing cisplatin to systemic anti-cancer therapy with reduced testicular toxicity and restored fertility.

Precision biomarker discovery powered by microscopy image fusion-assisted high spatial resolution ambient ionization mass spectrometry imaging.

Mass spectrometry imaging (MSI) using the ambient ionization technique enables a direct chemical investigation of biological samples with minimal sample pretreatment. However, detailed morphological information of the sample is often lost due to its limited spatial resolution. In this study, predictive high-resolution molecular imaging was produced by the fusion of ambient ionization MSI with optical microscopy of routine hematoxylin and eosin (H&E) staining. Specifically, desorption electrospray ionization (DESI) and nanospray desorption electrospray ionization (nanoDESI) mass spectrometry were employed to visualize lipid and protein species on mice tissue sections. The resulting molecular distributions obtained by ambient ionization MSI-microscopy fusion were verified with matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MSI and immunohistochemistry (IHC) staining. Label-free molecular imaging with 5-µm spatial resolution can be acquired using DESI and nanoDESI, whereas the typical spatial resolution of ambient ionization MSI was ∼100 µm. In this regard, sharpened molecular histology of tissue sections was achieved, providing complementary references to the pathology. Such a multi-modal integration enables the discovery of potential tumor biomarkers. After image fusion, more than a dozen potential biomarkers on a metastatic mouse lung tissue section and Luminal B breast tumor tissue section were identified.

Nucleotide-Dependent Bioautocatalytic Timer Reaction.

We describe a biochemical timer composed of three biocatalytic reactions involving three types of adenylate nucleotides: adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP). The timer is triggered by a small amount of ATP or ADP. An abrupt increase in the ATP concentration (following numerous amplification cycles) leads to a sudden increase of luminescence from the reaction mixture. The time point when the luminescence appears is found to be a function of the initial concentration of the triggering nucleotide (5.0 × 10(-8)-1.0 × 10(-6) M), even in the presence of a complex biological matrix. The mechanism of the observed dependence of the time of luminescence increase on the concentration has been confirmed with simple kinetic models. Due to the biocompatibility of the proposed trienzymatic reaction scheme (sensitivity to common nucleotides and occurrence in a neutral pH aqueous environment), the scheme can be used in bioengineered systems that require modulation of the response time (light emission) by concentration.

Low-level laser effects on simulated orthodontic tension side periodontal ligament cells.

OBJECTIVE: The purpose of this study was to analyze proliferation, inflammation, and osteogenic effects on periodontal ligament (PDL) cells after low-level laser therapy (LLLT) under simulated orthodontic tension conditions. BACKGROUND DATA: Low-level lasers affect fibroblast proliferation and collagen synthesis and reduce inflammation. Few studies have focused on the LLLT changes in the PDL caused by moving teeth. MATERIALS AND METHODS: A human PDL cell line was cultured in a -100 kPa tension incubator. The PDL cells were treated with a 670 nm low-level diode laser, output power of 500 mW (continuous wave modus) for 2.5 or 5 sec, spot area 0.25 cm(2), corresponding to 1.25 and 2.5 J at an energy density of 5 or 10 J/cm(2), respectively. PDL cell viability was assayed by detecting the ability of the cells to cleave tetrazolium salt to formazan dye. Inflammation and osteogenic markers were analyzed by Western blot analysis. RESULTS: PDL cell viablity increased in the experimental group, based on the ability of the cells to cleave tetrazolium salt at day 7 (p<0.05). The experimental group showed no difference in PDL cellular morphology compared with the control group. The inflammation markers inducible NO synthase (iNOS), cyclooxygenase (COX)-2 and interleukin (IL)-1 showed stronger expression in 5 and 10 J/cm(2) therapy at days 1 and 5, but decreased in expression at day 7. The osteogenic marker osteocalcin (OC) expression level was significantly higher at day 7 (p<0.05) than in the control cells. CONCLUSIONS: LLLT significantly increased PDL cell proliferation, decreased PDL cell inflammation, and increased PDL OC activity under the tension conditions used in this study.

Feasibility of a novel two-piece nasogastric feeding tube for patients with dysphagia.

INTRODUCTION: The exposed section of a traditional nasogastric (NG) tube can interfere with patients' social activities and thereby result in distress. This study was conducted to evaluate the feasibility and safety of a novel two-piece NG tube for patients with dysphagia. METHODS: Ten patients with dysphagia were recruited between November 2011 and May 2012. Patients who were unconscious or in critical condition, had a traditional NG tube < 50 cm or > 60 cm in fixed length, or were unable to follow instructions or sign consent forms were excluded. The two-piece NG tube, which was placed in the patients for one week, comprised a removable external tube that can be joined to an internal tube via a T-connector, which was placed close to the naris. Events related to safety (e.g. nasal pressure sores, number of unplanned extubation, displacement and spontaneous migration of the NG tube, other unpredictable injuries) and effectiveness (e.g. liquid food spills, tube obstruction, perfusion rate, other adverse circumstances) were assessed daily. RESULTS: All patients received feeding without complication using the two-piece NG tube and none experienced premature removal of the tube. No serious NG tube complications or malfunctions were observed. CONCLUSION: The results of this study indicate that the two-piece NG feeding tube is a feasible option for patients with dysphagia. Future improvements to the connector may help enhance its performance. A rigorous randomised controlled trial to examine the effects of the two-piece NG tube on patients' quality of life and quality of medical care is being planned.

Evaluation of human osteosarcoma cell line genotoxicity effects of mineral trixoide aggregate and calcium silicate cements.

INTRODUCTION: Mineral trioxide aggregate (MTA) and calcium silicate (CS) cements exhibit acceptable levels of cytocompatibility. The aim of the present study was to evaluate MTA and CS cement genotoxicity in a human osteosarcoma cell line (MG63). METHODS: A mitochondrial colorimetric assay was used to evaluate the MG63 survival rate. A DNA precipitation assay was used to detect the MG63 DNA damage after contact with MTA or CS cement extracts. RESULTS: The results showed that MTA and CS are cytocompatible with MG63 cells. There was no significant difference in the survival rate with MTA and CS materials (P > .05). Neither MTA nor CS cements causing MG63 cell DNA damage showed significant genotoxicity (P > .05). CONCLUSIONS: Both MTA and CS cements are compatible with MG63 cells, and they are not cancer-causing agents.

Expression of the inflammatory marker cyclooxygenase-2 in dental pulp cells cultured with mineral trioxide aggregate or calcium silicate cements.

INTRODUCTION: Mineral trioxide aggregate (MTA) and calcium silicate (CS) cements exhibit acceptable physical and chemical properties. The aim of the present study was to evaluate the effects of MTA and CS cements on inflammatory reactions in primary cultured human dental pulp cells. METHODS: The mitochondrial colorimetric assay was used to evaluate pulp cell survival rates. Fluorescent immunohistochemistry was used to observe focal adhesion kinase (FAK) and cyclooxygenase-2 (COX-2) distributions in the cells. Reverse transcription-polymerase chain reaction was used to assess COX-2 expression. RESULTS: The results showed that MTA and CS are biocompatible with pulp cells (P>.05). FAK was well-distributed in pulp cells in contact with both cements. Both MTA and CS cements induced pulp cell inflammation as evidenced by increased COX-2 expression. CONCLUSIONS: The present study demonstrated that MTA and CS cements are biocompatible with primary cultured pulp cells. Both cements can induce inflammatory COX-2 expression in the pulp cells.

Comparison of calcium and silicate cement and mineral trioxide aggregate biologic effects and bone markers expression in MG63 cells.

Bone cell (MG63) biocompatibility and bone marker expression were compared after calcium and silicate base cement (CS) and mineral trioxide aggregate (MTA) treatment. X-ray diffraction was used to identify material surface structure, and tetrazolium bromide colorimetric assay was used to evaluate the cell viability. The relative mitogen activation protein kinase expression was compared with Western blot, and bone marker expression was evaluated with reverse transcriptase polymerization chain reaction. The results showed that CS and MTA are similar chemical structures and biocompatible with MG63 cells. CS and MTA cements showed good MG63 cell proliferation by high phosphor extracellular signal-regulated kinase expression levels. CS and MTA cements showed the evident type I collagen, osteocalcin, alkaline phosphatase, bone sialoprotein, and osteopontin expression. Both MTA and CS cements are biocompatible and appear to have osetoconduction effects on bone cells.