In situ metabolomic analysis of osteonecrosis of the femoral head (ONFH) using MALDI MSI.

Osteonecrosis of the femoral head (ONFH) is a common orthopedic disease characterized by disability and deformity. To better understand ONFH at molecular level and to explore the possibility of early diagnosis, instead of diagnosis based on macroscopic spatial characteristics, a matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) method was developed for ONFH disease for the first time. The most challenging step for ONFH MSI is to deal with human bone tissues which are much harder than the other biological samples studied by the reported MSI studies. In this work, the MSI sectioning method of hard bone tissues was established using tender acids and a series of test criteria. Small-molecule metabolites, such as lipids and amino acids, were detected in bone sections, realizing the in situ detection of spatial distribution of biometabolites. By comparing the distribution of metabolites from different regions of normal femoral head, ONFH bone tissue (ONBT), and adjacent ONFH bone tissue (ANBT), the whole process of femoral head from normal stage to necrosis was monitored and visualized at molecular level. Moreover, this developed MSI method was used for metabolomics study of ONFH. 72 differential metabolites were identified, suggesting that disturbances in energy metabolism and lipid metabolism affected the normal life activities of osteoblasts and osteoclasts. This study provides new perspectives for future pathological studies of ONFH.

Ants may buffer the Janzen-Connell effect in a tropical forest in Southwest China.

Mutualistic symbioses between ants and plants are widespread in nature. Ants can deter unwanted pests and provide protection for plants in return for food or housing rewards. Using a long-term demographic dataset in a tropical seasonal rain forest in Southwest China, we found that associations with ants positively influenced seedling survival and adult growth, and also, species with extrafloral nectaries experienced weaker conspecific negative density dependence compared with species without extrafloral nectaries. Furthermore, we found strong evidence suggesting that species in our forest experienced conspecific density dependence, which we interpreted as heavy pest pressure that may drive the development of anti-pest symbioses such as the plant-ant relationship. Our findings suggest that ants and conspecific neighbors play important but inverse roles on plant survival and growth and that ants can buffer tree neighborhood interactions in this tropical forest.

Recent advances in mass spectrometry imaging of single cells.

Mass spectrometry imaging (MSI) is a sensitive, specific, label-free imaging analysis technique that can simultaneously obtain the spatial distribution, relative content, and structural information of hundreds of biomolecules in cells and tissues, such as lipids, small drug molecules, peptides, proteins, and other compounds. The study of molecular mapping of single cells can reveal major scientific issues such as the activity pattern of living organisms, disease pathogenesis, drug-targeted therapy, and cellular heterogeneity. Applying MSI technology to the molecular mapping of single cells can provide new insights and ideas for the study of single-cell metabolomics. This review aims to provide an informative resource for those in the MSI community who are interested in single-cell imaging. Particularly, we discuss advances in imaging schemes and sample preparation, instrumentation improvements, data processing and analysis, and 3D MSI over the past few years that have allowed MSI to emerge as a powerful technique in the molecular imaging of single cells. Also, we highlight some of the most cutting-edge studies in single-cell MSI, demonstrating the future potential of single-cell MSI. Visualizing molecular distribution at the single-cell or even sub-cellular level can provide us with richer cell information, which strongly contributes to advancing research fields such as biomedicine, life sciences, pharmacodynamic testing, and metabolomics. At the end of the review, we summarize the current development of single-cell MSI technology and look into the future of this technology.

Effects of personal protective clothing on firefighters' gait analyzed using a three-dimensional motion capture system.

Objectives. The effects of personal protective clothing (PPC) on firefighters' gait were investigated to develop high-performance PPC. Methods. Thirteen participants participated in human trials with three types of PPC (firefighter protective clothing [FPC], semi-enclosed chemical protective clothing [CPC_semi] and fully enclosed chemical protective clothing [CPC_full]) and a T-shirt (control clothing [CON]). A three-dimensional (3D) motion capture system was used to obtain gait parameters (step length, step width, stride frequency, gait speed and toe-out angle) and the range of motion (ROM) of the joints (hip, knee and ankle). Results. PPC produced an increase in step width (23.4%, p > 0.05), but the gait speed (9.1%) and stride frequency (6.4%) decreased compared with the CON results. ROM is affected by the PPC type and joint. FPC and CPC_semi had no significant effect in terms of ROM of the hip and knee besides the landing angle of the knee. However, CPC_full had a significant effect on the maximum extension angle of the hip and maximum flexion angle of the knee, which reached up to 27.2%. Conclusion. The ROM of the firefighter's lower limbs was limited by PPC. This study offers insights into next-generation PPC design and development, as well as guidelines for training and firefighting.

Tracing the migration and transformation of metabolites in xylem during wood growth by mass spectrometry imaging.

Metabolites in the xylem experience several migration and transformation processes during tree growth. Their composition and distributions can reflect the environment that the wood lived through. Herein, a matrix-assisted laser desorption/ionization mass spectrometry imaging method was developed to investigate the migration and transformation of metabolites in the xylem during heartwood formation and after mechanical injury. The thickness of the wood slice, the type of matrix and its manner of deposition were optimized to improve ionization response and spatial resolution. The mass difference correlation (MDC) data processing method was proposed to improve the efficiency of compound identification, in which the compounds were classified by their molecular weight. The compound species was identified by results calculated using MDC and the experimental results from MS/MS. The directly identified metabolites, whose type and number were found to be quite different between sapwood and heartwood, demonstrated the transformation and migration of metabolites from sapwood to heartwood. Additionally, two kinds of resins produced from different positions were identified by MSI simultaneously, even though their heterogeneous distribution was not visible in optical images. The origin and type of the two resins were deduced from the identified compounds and their molecular distribution. This work provides a method to directly reveal metabolite migration and transformation mechanisms in xylem during wood growth.

Catalytic asymmetric nucleophilic fluorination using BF3·Et2O as fluorine source and activating reagent.

Fluorination using chiral catalytic methods could result in a direct access to asymmetric fluorine chemistry. However, challenges in catalytic asymmetric fluorinations, especially the longstanding stereochemical challenges existed in BF3·Et2O-based fluorinations, have not yet been addressed. Here we report the catalytic asymmetric nucleophilic fluorination using BF3·Et2O as the fluorine reagent in the presence of chiral iodine catalyst. Various chiral fluorinated oxazine products were obtained with good to excellent enantioselectivities (up to >99% ee) and diastereoselectivities (up to >20:1 dr). Control experiments (the desired fluoro-oxazines could not be obtained when Py·HF or Et3N·3HF were employed as the fluorine source) indicated that BF3·Et2O acted not only as a fluorine reagent but also as the activating reagent for activation of iodosylbenzene.

The asymmetric construction of CF3-containing spiro-thiazolone-pyrrolidine compounds via [3 + 2] cycloaddition.

The first organocatalytic method for the asymmetric construction of CF3-containing spiro-thiazolone-pyrrolidine compounds has been developed. This is one of the very few methods to reach spiro-thiazolones, and afforded the products in excellent yields and stereoselectivities catalysed by only 1 mol% cinchona alkaloid-derived catalyst.

Comparison of heavy metal accumulation and cadmium phytoextraction rates among ten leading tobacco (Nicotiana tabacum L.) cultivars in China.

Cadmium (Cd) contamination is one of the most serious global environmental problems, and phytoremediation, which uses Cd-accumulator plants, is potentially one of the sustainable solutions. Pot experiments with natural and Cd-amended soils were conducted to investigate the accumulation of heavy metals in 10 leading cultivars of tobacco in China. The extraction ability and profiles of Cd accumulation among plant organs were also analyzed. The tobacco roots accumulated cobalt, nickel, and Cd, while the leaf highly bioaccumulated Cd and lowly accumulated zinc, selenium and mercury. The transport from the tobacco stem to the leaf plays a critical role in the accumulation of these elements. The ratios of Cd concentration in the leaves at lower, middle and upper positions were comparatively stable. The high Cd-extracting cultivars were "Hongda", "NC89" and "Zhongyan 100" when grown in normal soils, "CuiBi 1" and "Hongda" in moderately contaminated soils, and "YuYan 87", "LongJiang 851" and "K326" in severely contaminated soils. Tobacco leaves could accumulate about 80% of the total Cd extracted from the soil by the plant. Considering the Cd-extraction limitations exhibited by leading tobacco cultivars, screening of germplasm resources for high or low levels of Cd-accumulation is still an important target for the future.

Cross-Subtype Detection of HIV-1 Capsid p24 Antigen Using a Sensitive Europium Nanoparticle Assay.

Accurate and early detection of diverse HIV-1 subtypes using currently available p24 antigen assays have been a major challenge. We report the development of a sensitive time resolved fluorescence (TRF) europium nanoparticle immuno assay for cross subtype detection of p24 antigen using broadly cross-reactive antibodies. Several antibodies were tested for optimal reactivity with antigens of diverse HIV-1 subtypes and circulating recombinant forms. We tested HIV strains using this assay for sensitivity and quantification ability at the pico-gram per millilter level. We identified two broadly cross-reactive HIV-1 p24 antibodies C65690M and ANT-152, which detected all strains of HIV tested. These two antibodies also yielded a better signal to cutoff ratio for the same amount of antigen tested in comparison to a commercial assay. Using an appropriate combination of C65690M and ANT-152 p24 antibodies capable of detecting all HIV types and highly sensitive TRF-based europium nano particle assay platform, we developed a sensitive p24 antigen assay that can detect HIV infection of all HIV subtypes and may be useful in early detection.

Sensitive Detection and Simultaneous Discrimination of Influenza A and B Viruses in Nasopharyngeal Swabs in a Single Assay Using Next-Generation Sequencing-Based Diagnostics.

Reassortment of 2009 (H1N1) pandemic influenza virus (pdH1N1) with other strains may produce more virulent and pathogenic forms, detection and their rapid characterization is critical. In this study, we reported a "one-size-fits-all" approach using a next-generation sequencing (NGS) detection platform to extensively identify influenza viral genomes for diagnosis and determination of novel virulence and drug resistance markers. A de novo module and other bioinformatics tools were used to generate contiguous sequence and identify influenza types/subtypes. Of 162 archived influenza-positive patient specimens, 161(99.4%) were positive for either influenza A or B viruses determined using the NGS assay. Among these, 135(83.3%) were A(H3N2), 14(8.6%) were A(pdH1N1), 2(1.2%) were A(H3N2) and A(pdH1N1) virus co-infections and 10(6.2%) were influenza B viruses. Of the influenza A viruses, 66.7% of A(H3N2) viruses tested had a E627K mutation in the PB2 protein, and 87.8% of the influenza A viruses contained the S31N mutation in the M2 protein. Further studies demonstrated that the NGS assay could achieve a high level of sensitivity and reveal adequate genetic information for final laboratory confirmation. The current diagnostic platform allows for simultaneous identification of a broad range of influenza viruses, monitoring emerging influenza strains with pandemic potential that facilitating diagnostics and antiviral treatment in the clinical setting and protection of the public health.

Sensitive detection of influenza viruses with Europium nanoparticles on an epoxy silica sol-gel functionalized polycarbonate-polydimethylsiloxane hybrid microchip.

In an effort to develop new tools for diagnosing influenza in resource-limited settings, we fabricated a polycarbonate (PC)-polydimethylsiloxane (PDMS) hybrid microchip using a simple epoxy silica sol-gel coating/bonding method and employed it in sensitive detection of influenza virus with Europium nanoparticles (EuNPs). The incorporation of sol-gel material in device fabrication provided functionalized channel surfaces ready for covalent immobilization of primary antibodies and a strong bonding between PDMS substrates and PC supports without increasing background fluorescence. In microchip EuNP immunoassay (µENIA) of inactivated influenza viruses, replacing native PDMS microchips with hybrid microchips allowed the achievement of a 6-fold increase in signal-to-background ratio, a 12-fold and a 6-fold decreases in limit-of-detection (LOD) in influenza A and B tests respectively. Using influenza A samples with known titers, the LOD of influenza µENIA on hybrid microchips was determined to be ~10(4) TCID50 titer/mL and 10(3)-10(4) EID50 titer/mL. A comparison test indicated that the sensitivity of influenza µENIA enhanced using the hybrid microchips even surpassed that of a commercial laboratory influenza ELISA test. In addition to the sensitivity improvement, assay variation was clearly reduced when hybrid microchips instead of native PDMS microchips were used in the µENIA tests. Finally, infectious reference viruses and nasopharyngeal swab patient specimens were successfully tested using µENIA on hybrid microchip platforms, demonstrating the potential of this unique microchip nanoparticle assay in clinical diagnosis of influenza. Meanwhile, the tests showed the necessity of using nucleic acid confirmatory tests to clarify ambiguous test results obtained from prototype or developed point-of-care testing devices for influenza diagnosis.

Current Approaches for Diagnosis of Influenza Virus Infections in Humans.

Despite significant advancement in vaccine and virus research, influenza continues to be a major public health concern. Each year in the United States of America, influenza viruses are responsible for seasonal epidemics resulting in over 200,000 hospitalizations and 30,000-50,000 deaths. Accurate and early diagnosis of influenza viral infections are critical for rapid initiation of antiviral therapy to reduce influenza related morbidity and mortality both during seasonal epidemics and pandemics. Several different approaches are currently available for diagnosis of influenza infections in humans. These include viral isolation in cell culture, immunofluorescence assays, nucleic acid amplification tests, immunochromatography-based rapid diagnostic tests, etc. Newer diagnostic approaches are being developed to overcome the limitations associated with some of the conventional detection methods. This review discusses diagnostic approaches currently available for detection of influenza viruses in humans.

Novel Time-Resolved Fluorescence Europium Nanoparticle Immunoassay for Detection of Human Immunodeficiency Virus-1 Group O Viruses Using Microplate and Microchip Platforms.

Accurate detection and quantification of HIV-1 group O viruses have been challenging for currently available HIV assays. We have developed a novel time-resolved fluorescence (TRF) europium nanoparticle immunoassay for HIV-1 group O detection using a conventional microplate enzyme-linked immunosorbent assay (ELISA) and a microchip platform. We screened several antibodies for optimal reactivity with several HIV-1 group O strains and identified antibodies that can detect all the strains of HIV-1 group O that were available for testing. The antibodies were used to develop a conventional ELISA format assay and an in-house developed europium nanoparticle-based assay for sensitivity. The method was evaluated on both microwell plate and microchip platforms. We identified two specific and sensitive antibodies among the six we screened. The antibodies, C65691 and ANT-152, were able to quantify 15 and detect all 17 group O viruses, respectively, as they were broadly cross-reactive with all HIV-1 group O strains and yielded better signals compared with other antibodies. We have developed a sensitive assay that reflects the actual viral load in group O samples by using an appropriate combination of p24 antibodies that enhance group O detection and a highly sensitive TRF-based europium nanoparticle for detection. The combination of ANT-152 and C65690M in the ratio 3:1 was able to give significantly higher signals in our europium-based assay compared with using any single antibody.

Nanomicroarray and multiplex next-generation sequencing for simultaneous identification and characterization of influenza viruses.

Conventional methods for detection and discrimination of influenza viruses are time consuming and labor intensive. We developed a diagnostic platform for simultaneous identification and characterization of influenza viruses that uses a combination of nanomicroarray for screening and multiplex next-generation sequencing (NGS) assays for laboratory confirmation. The nanomicroarray was developed to target hemagglutinin, neuraminidase, and matrix genes to identify influenza A and B viruses. PCR amplicons synthesized by using an adapted universal primer for all 8 gene segments of 9 influenza A subtypes were detected in the nanomicroarray and confirmed by the NGS assays. This platform can simultaneously detect and differentiate multiple influenza A subtypes in a single sample. Use of these methods as part of a new diagnostic algorithm for detection and confirmation of influenza infections may provide ongoing public health benefits by assisting with future epidemiologic studies and improving preparedness for potential influenza pandemics.

A highly sensitive europium nanoparticle-based immunoassay for detection of influenza A/B virus antigen in clinical specimens.

We report the development of a novel europium nanoparticle-based immunoassay (ENIA) for rapid detection of influenza A and influenza B viruses. The ENIA demonstrated sensitivities of 90.7% (147/162) for influenza A viruses and 81.80% (9/11) for influenza B viruses compared to those for an in-house reverse transcription (RT)-PCR assay in testing of influenza-positive clinical samples.

Development of a microchip Europium nanoparticle immunoassay for sensitive point-of-care HIV detection.

Rapid, sensitive and specific diagnostic assays play an indispensable role in determination of HIV infection stages and evaluation of efficacy of antiretroviral therapy. Recently, our laboratory developed a sensitive Europium nanoparticle-based microtiter-plate immunoassay capable of detecting target analytes at subpicogram per milliliter levels without the use of catalytic enzymes and signal amplification processes. Encouraged by its sensitivity and simplicity, we continued to miniaturize this assay to a microchip platform for the purpose of converting the benchtop assay technique to a point-of-care test. It was found that detection capability of the microchip platform could be readily improved using Europium nanoparticle probes. We were able to routinely detect 5 pg/mL (4.6 attomoles) of HIV-1 p24 antigen at a signal-to-blank ratio of 1.5, a sensitivity level reasonably close to that of microtiter-plate Europium nanoparticle assay. Meanwhile, use of the microchip platform effectively reduced sample/reagent consumption 4.5 fold and shortened total assay time 2 fold in comparison with microtiter plate assays. Complex matrix substance in plasma negatively affected the microchip assays and the effects could be minimized by diluting the samples before loading. With further improvements in sensitivity, reproducibility, usability, assay process simplification, and incorporation of portable time-resolved fluorescence reader, Europium nanoparticle immunoassay technology could be adapted to meet the challenges of point-of-care diagnosis of HIV or other health-threatening pathogens at bedside or in resource-limited settings.

Nanoparticle-functionalized porous polymer monolith detection elements for surface-enhanced Raman scattering.

The use of porous polymer monoliths functionalized with silver nanoparticles is introduced in this work for high-sensitivity surface-enhanced Raman scattering (SERS) detection. Preparation of the SERS detection elements is a simple process comprising the synthesis of a discrete polymer monolith section within a silica capillary, followed by physically trapping silver nanoparticle aggregates within the monolith matrix. A SERS detection limit of 220 fmol for Rhodamine 6G is demonstrated, with excellent signal stability over a 24 h period. The capability of the SERS-active monolith for label-free detection of biomolecules was demonstrated by measurements of bradykinin and cytochrome c. The SERS-active monoliths can be readily integrated into miniaturized micrototal-analysis systems for online and label-free detection for a variety of biosensing, bioanalytical, and biomedical applications.

Flow-through immunosensors using antibody-immobilized polymer monoliths.

High-sensitivity and rapid flow-through immunosensors based on photopolymerized surface-reactive polymer monoliths are investigated. The porous monoliths were synthesized within silica capillaries from glycidyl methacrylate and ethoxylated trimethylolpropane triacrylate precursors, providing a tortuous pore structure with high surface area for the immobilization of antibodies or other biosensing ligands. The unique morphology of the monolith ensures efficient mass transport and interactions between solvated analyte molecules and covalently immobilize antibodies anchored to the monolith surface, resulting in rapid immunorecognition. The efficacy of this approach is demonstrated through a direct immunoassay model using anti-IgG as a monolith-bound capture antibody and fluorescein-labeled IgG as an antigen. In situ antigen measurements exhibited a linear response over a concentration range between 0.1 and 50 ng/mL with 5 min assay times, while controllable injection of 1 µL volumes of antigen through the monolith elements yielded a mass detection limit of 100 pg ((∼700amol). These results suggest that porous monolith supports represent a flexible and promising material for the fabrication of rapid and sensitive immunosensors suitable for integration into capillary or microfluidic devices.

Mixed-mode electrokinetic and chromatographic peptide separations in a microvalve-integrated polymer chip.

A cycloolefin polymer chip supporting the concatenation of isoelectric focusing (IEF) and reversed-phase liquid chromatography (RPLC) is demonstrated for high throughput two dimensional peptide separations. A unique benefit of the mixed-mode platform is the ability of IEF to act as a highly concentrating electrokinetic separation mode for effective isolation of sample components prior to RPLC. The thermoplastic chip contains integrated high pressure microvalves, enabling uniform sample transfer from the IEF channel to multiple parallel RPLC channels, gradient elution from each RPLC column, and hydrodynamic isolation between the separation dimensions. The reusable system is shown to provide efficient 2-D separations together with facile interfacing with MALDI-MS, suggesting a new path towards effective peptide analysis from complex samples.

High-pressure on-chip mechanical valves for thermoplastic microfluidic devices.

A facile method enabling the integration of elastomeric valves into rigid thermoplastic microfluidic chips is described. The valves employ discrete plugs of elastomeric polydimethylsiloxane (PDMS) integrated into the thermoplastic substrate and actuated using a threaded stainless steel needle. The fabrication process takes advantage of poly(ethylene glycol) (PEG) as a sacrificial molding material to isolate the PDMS regions from the thermoplastic flow channels, while yielding smooth contact surfaces with the PDMS valve seats. The valves introduce minimal dead volumes, and provide a simple mechanical means to achieve reproducible proportional valving within thermoplastic microfluidic systems. Burst pressure tests reveal that the valves can withstand pressures above 12 MPa over repeated open/close cycles without leakage, and above 24 MPa during a single use, making the technology well suited for applications such as high performance liquid chromatography. Proportional valve operation is demonstrated using a multi-valve chemical gradient generator fabricated in cyclic olefin polymer.