Analysis of Glycopeptides by HPLC MS and Detection of Leukemic Cells by Microfluidics using Time Delayed Integration Spectral Flow Cytometry

Protein glycosylation drives many biological processes and serves as marker for disease; therefore, the development of tools to study glycosylation is an essential and growing area of research. Mass spectrometry can be used to identify both the glycans of interest and the glycosylation sites to which those glycans are attached, when proteins are proteolytically digested and their glycopeptides are analyzed by a combination of high-resolution mass spectrometry (MS) and tandem mass spectrometry (MS/MS) methods. One major challenge in these experiments is collecting the requisite MS/MS data. The digested glycopeptides are often present in complex mixtures and in low abundance, and the most commonly used approach to collect MS/MS data on these species is data-dependent acquisition (DDA), where only the most intense precursor ions trigger MS/MS. DDA results in limited glycopeptide coverage. Semi-targeted data acquisition is an alternative experimental approach that can alleviate this difficulty. However, due to the massive heterogeneity of glycopeptides, it is not obvious how to expediently generate inclusion lists for these types of analyses. To solve this problem, we developed the software tool GlycoPep MassList, which can be used to generate inclusion lists for liquid chromatography tandem-mass spectrometry (LC-MS/MS) experiments. The utility of the software was tested by conducting comparisons between semi-targeted and untargeted data-dependent analysis experiments on a variety of proteins, including IgG, a protein whose glycosylation must be characterized during its production as a biotherapeutic. When the GlycoPep MassList software was used to generate inclusion lists for LC-MS/MS experiments, more unique glycopeptides were selected for fragmentation. Generally, ∼30 % more unique glycopeptides can be analyzed per protein, in the simplest cases, with low background. In cases where background ions from proteins or other interferents are high, usage of an inclusion list is even more advantageous. The software is free and publicly accessible. In another research project, we describe a unique flow cytometer (TDI SFC) that combines the high spectral resolution of spectral flow cytometry (SFC) with a CCD operated in time-delayed integration (TDI) mode for the automated immunophenotyping of rare, low abundant cells. A microfluidic device providing 1-D focusing was used to sheath cells through a 488 nm laser excitation beam. Using epi-illumination, a spectrograph was included into the emission optical path to spectrally disperse the emission along one axis of a CCD camera. The parallel shift rate of the CCD was synchronized to the cell travel through the field-of-view, which was defined by the excitation volume. This TDI SFC format allowed the CCD shutter to remain open during signal acquisition and as such, the duty cycle was ~100% allowing for rare cells to not be missed. Fluorescent calibration beads were used to optimize synchronization of the CCD’s TDI clocking with the sheathed cell velocity, TDI SFC sensitivity, excitation power intensity, epi-illumination objective’s numerical aperture, and total integration time. TDI integrated signals of 106 counts at a signal-to-noise ratio (SNR) of 610 for beads corresponding to a load of 4×105 antibodies per bead was achieved. Additionally, we evaluated the multiplexing capabilities by performing spectral deconvolution. Finally, a proof-of-concept application was undertaken to immunophenotype rare cells, specifically leukemic cells circulating in the blood of patients with B-cell acute lymphoblastic leukemia (B-ALL) for monitoring measurable residual disease (MRD). A B-ALL cell line was stained against a leukemic marker (TdT) to successfully discriminate TdT(+) circulating leukemic cells from normal B cells at very low cell counts (≤100 cells)

Development and characterisation of a copper-based oxygen carrier for chemical-looping with oxygen uncoupling (CLOU)

In chemical-looping, a fuel is oxidised by a solid metal oxide, MeO, in one reactor: (2$n+m$)MeO+C$_{n}$H$_{2m}\rightarrow$(2$n+m$)Me+$m$H$_{2}$O+$n$CO$_{2}$. The exit gas yields pure CO$_{2}$ after the steam has been condensed. The reduced metal oxide, Me, is transferred to an oxidation reactor and regenerated: Me+air$\rightarrow$MeO. Adding these reactions, the fuel has been combusted, but the CO$_{2}$ has been separated from the nitrogen in air. In fact, it is in a suitable form for sequestration in the Earth where prevention of greenhouse gas emissions to the atmosphere is desired. Generally, Me is a transition metal and, to withstand many such redox cycles, it has to be supported on a suitable refractory oxide, with particles of the resulting construct being termed the "oxygen carrier". This Dissertation is concerned with the release and uptake of gaseous oxygen when Me is copper. In particular, the interest is in the following reaction at temperatures exceeding ~900°C undertaken in a fluidised bed reactor:$\\$ 4CuO$_{(s)}\Leftrightarrow$2Cu$_{2}$O$_{(s)}$+O$_{2(g)}$. (1)$\\$ The value of this reaction is that the oxygen released as part of a chemical looping scheme is important in combusting unreactive solid fuels, e.g. coal chars, whilst the Cu$_{2}$O could, in principle, be further reduced to Cu by the more reactive components of the fuel. This Dissertation investigates the development and characterisation of suitable, Cu-based oxygen carriers, which must (i) be inexpensive and easy to produce at a large scale and (ii) remain stable in prolonged operation in terms of mechanical integrity and chemical reactivity when fluidised. Here, a suitable oxygen carrier was developed, satisfying the above criteria, using a wet-mixing method and containing nominally 60 wt% CuO, 23 wt% Al$_{2}$O$_{3}$ and 17 wt% CaO. In particular, it was found that this oxygen carrier could operate between CuO and Cu$_{2}$O without problem in a circulating fluidised bed but agglomeration and de-fluidisation was observed when the carrier was re-oxidised from the Cu form. For design, it is important to understand the thermodynamics and kinetics of the release of gaseous O$_{2}$ from the oxygen carrier, because the combustion of the solid fuel depends critically on this reaction. A novel method was developed to measure experimentally the thermodynamics of reaction (1) for the supported copper oxide. It was found that the thermodynamic equilibrium deviated slightly from that of the pure CuO/Cu$_{2}$O system reported in the literature and that the enthalpy of reaction was lower by ~ 15%; the reasons for this are discussed. The rate of release of O$_{2}$ from the oxygen carrier was investigated using a thermogravimetric analyser and the activation energy for the forward reaction of (1) was found to be 59.7$\pm$5.6 kJ/mol, obtained after appropriate modelling of the external mass transfer resistances present in the experimental apparatus. A critical analysis of the seemingly disparate activation energies reported in the literature revealed that the activation energy of the forward step in reaction (1) was, in fact, similar for many CuO-based oxygen carriers supported on different materials. The associated pre-exponential factor for the forward rate constant was also determined in the present research, and the kinetic parameters were used in a numerical model to predict the behaviour of the oxygen carriers in a fluidised bed reactor. Excellent agreement between theory and experiment was found, confirming that the kinetic parameters obtained in this work reflect the intrinsic chemical kinetics of the oxygen carrier, rather than being totally dominated by transport effects

Time-Delayed Integration–Spectral Flow Cytometer (TDI-SFC) for Low-Abundance-Cell Immunophenotyping

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Analytical Chemistry, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://doi.org/10.1021/acs.analchem.9b00021.We describe a unique flow cytometer (TDI-SFC) for the immunophenotyping of low-abundance cells, particularly when cell counts are sample-limited and operationally difficult for analysis by fluorescence microscopy (>100 cells) or multiparameter flow cytometry (MFC, <10 000 cells). TDI-SFC combines the high spectral resolution of spectral flow cytometry (SFC) with a CCD operated in time-delayed integration (TDI) for improved duty cycle and sensitivity. Cells were focused with a 1D-sheathing microfluidic device, and fluorescence emission generated from a 488 nm laser was collected by epi-illumination and dispersed along one axis of a CCD by a spectrograph. Along the other axis, the CCD’s shift rate was clocked at a rate that closely matched the cells’ velocity through the field of view. This TDI-SFC format allowed the CCD shutter to remain open during signal acquisition, providing a duty cycle ∼100% and assurance that ∼95% cells were interrogated. We used fluorescent beads to optimize synchronization of TDI clocking with the sheathed-cell velocity and to improve sensitivity via the excitation intensity, epi-illumination numerical aperture, and integration time. TDI achieved integrated signals of 106 counts at a signal-to-noise ratio (SNR) of 610 for beads corresponding to a load of 4 × 105 antibodies. We also evaluated multiplexing capabilities by spectral deconvolution and undertook a proof-of-concept application to immunophenotype low-abundance cells; the demonstration consisted of immunophenotyping a model cell line, in this case SUP-B15 cells representing B-cell acute lymphoblastic leukemia (B-ALL). The B-ALL cell line was stained against a leukemic marker (terminal deoxynucleotidyl transferase, TdT), and we successfully used spectral unmixing to discriminate TdT(+) cells from TdT(−) cells even at low cell counts (∼100 cells). The TDI-SFC could potentially be used in any application requiring the immunophenotyping of low-abundance cells, such as in monitoring measurable residual disease in acute leukemias following affinity enrichment of circulating leukemia cells from peripheral blood

GlycoPep MassList: Software to Generate Massive Inclusion Lists for Glycopeptide Analyses

Protein glycosylation drives many biological processes and serves as markers for disease; therefore, the development of tools to study glycosylation is an essential and growing area of research. Mass spectrometry can be used to identify both the glycans of interest and the glycosylation sites to which those glycans are attached, when proteins are proteolytically digested and their glycopeptides are analyzed by a combination of high-resolution mass spectrometry (MS) and tandem mass spectrometry (MS/MS) methods. One major challenge in these experiments is collecting the requisite MS/MS data. The digested glycopeptides are often present in complex mixtures and in low abundance, and the most commonly used approach to collect MS/MS data on these species is data-dependent acquisition (DDA), where only the most intense precursor ions trigger MS/MS. DDA results in limited glycopeptide coverage. Semi-targeted data acquisition is an alternative experimental approach that can alleviate this difficulty. However, due to the massive heterogeneity of glycopeptides, it is not obvious how to expediently generate inclusion lists for these types of analyses. To solve this problem, we developed the software tool GlycoPep MassList, which can be used to generate inclusion lists for liquid chromatography tandem-mass spectrometry (LC-MS/MS) experiments. The utility of the software was tested by conducting comparisons between semi-targeted and untargeted data-dependent analysis experiments on a variety of proteins, including IgG, a protein whose glycosylation must be characterized during its production as a biotherapeutic. When the GlycoPep MassList software was used to generate inclusion lists for LC-MS/MS experiments, more unique glycopeptides were selected for fragmentation. Generally, ∼30 % more unique glycopeptides can be analyzed per protein, in the simplest cases, with low background. In cases where background ions from proteins or other interferents are high, usage of an inclusion list is even more advantageous. The software is freely publically accessible

Study on the Inhibitory Effects of Ephedra Aconite Asarum

Dendritic cells (DCs) can secrete cytokines stimulated by lipopolysaccharide (LPS), which leads to not just acute inflammatory responses but also Th1 polarization. Furtherly, chronic inflammation or autoimmune diseases could be triggered. As a classic Traditional Chinese Medicine formula, Ephedra Aconite Asarum Decoction with the main ingredients of ephedrine and hypaconitine can show effect on anti-inflammation and immunoregulation. But it remains unclear whether Ephedra Aconite Asarum Decoction controls DCs. In this study, we investigated the effects of Ephedra Aconite Asarum Decoction on LPS-induced bone marrow-derived DCs (BMDCs) in vitro. We found that Ephedra Aconite Asarum Decoction lowered surface costimulators on DCs and reduced the expression of Th1 type cytokines. Yet it is slightly beneficial for shifting to Th2. Our work reveals that the Ephedra Aconite Asarum Decoction can regulate Th1 inflammation through intervening DCs

Using proper orthogonal decomposition to solve heat transfer process in a flat tube bank fin heat exchanger

Proper orthogonal decomposition (POD) reduced-order model can save computing time by reducing the dimension of physical problems and reconstructing physical fields. It is especially suitable for large-scale complex problems in engineering, such as ground heat utilization, sea energy development, mineral exploitation, multiphase flow and flow and heat transfer with complex structure. In this paper, the POD reduced-order model was used to calculate the heat transfer in a flat tube bank fin heat exchanger. The calculating results of the finite volume method (FVM) were adopted as the snapshot samples. Singular value decomposition method was used to decompose the samples to obtain a series of bases and corresponding coefficients on sampling conditions. With these coefficients, interpolation method was used to calculate the coefficients on predicting conditions. And the physical field has been reconstructed using the bases and the interpolated coefficients directly. In the calculation of heat transfer unit of flat tube fin heat exchanger, air-side Reynolds number, transverse tube spacing and the fin spacing were chosen as the variables. The results obtained by the POD method are in good agreement with the results calculated by the FVM. Moreover, the POD reduced-order model presented in this paper is more advantageous in comparison with the FVM in terms of accuracy, suitability, and computational speed.Cited as: Wang, Y., Xia, X., Wang, Y., et al. Using proper orthogonal decomposition to solve heat transfer process in a flat tube bank fin heat exchanger. Advances in Geo-Energy Research, 2017, 1(3): 158-170, doi: 10.26804/ager.2017.03.0

Data integrity auditing without private key storage for secure cloud storage

Using cloud storage services, users can store their data in the cloud to avoid the expenditure of local data storage and maintenance. To ensure the integrity of the data stored in the cloud, many data integrity auditing schemes have been proposed. In most, if not all, of the existing schemes, a user needs to employ his private key to generate the data authenticators for realizing the data integrity auditing. Thus, the user has to possess a hardware token (e.g. USB token, smart card) to store his private key and memorize a password to activate this private key. If this hardware token is lost or this password is forgotten, most of the current data integrity auditing schemes would be unable to work. In order to overcome this problem, we propose a new paradigm called data integrity auditing without private key storage and design such a scheme. In this scheme, we use biometric data (e.g. iris scan, fingerprint) as the user's fuzzy private key to avoid using the hardware token. Meanwhile, the scheme can still effectively complete the data integrity auditing.We utilize a linear sketch with coding and error correction processes to confirm the identity of the user. In addition, we design a new signature scheme which not only supports blockless verifiability, but also is compatible with the linear sketch. The security proof and the performance analysis show that our proposed scheme achieves desirable security and efficiency

Nadir oxygen delivery is associated with postoperative acute kidney injury in low-weight infants undergoing cardiopulmonary bypass

BackgroundAcute kidney injury (AKI) is common after cardiac surgery with cardiopulmonary bypass (CPB) and is associated with increased mortality and morbidity. Nadir indexed oxygen delivery (DO2i) lower than the critical threshold during CPB is a risk factor for postoperative AKI. The critical DO2i for preventing AKI in children has not been well studied. The study aimed to explore the association between nadir DO2i and postoperative AKI in infant cardiac surgery with CPB.MethodsFrom August 2021 to July 2022, 413 low-weight infants (≤10 kg) undergoing cardiac surgery with CPB were consecutively enrolled in this prospective observational study. Nadir DO2i was calculated during the hypothermia and rewarming phases of CPB, respectively. The association between nadir DO2i and postoperative AKI was investigated in mild hypothermia (32–34°C) and moderate hypothermia (26–32°C).ResultsA total of 142 (38.3%) patients developed postoperative AKI. In patients undergoing mild hypothermia during CPB, nadir DO2i in hypothermia and rewarming phases was independently associated with postoperative AKI. The cutoff values of nadir DO2i during hypothermia and rewarming phases were 258 mL/min/m2 and 281 mL/min/m2, respectively. There was no significant association between nadir DO2i and postoperative AKI in patients undergoing moderate hypothermia during CPB.ConclusionIn low-weight infants undergoing mild hypothermia during CPB, the critical DO2i for preventing AKI was 258 mL/min/m2 in the hypothermia phase and 281 mL/min/m2 for rewarming. Moreover, an individualized critical DO2i threshold should be advocated during CPB

Role of Bile Acids in Bariatric Surgery

Bariatric surgery has been proved to be effective and sustainable in the long-term weight-loss and remission of metabolic disorders. However, the underlying mechanisms are still far from fully elucidated. After bariatric surgery, the gastrointestinal tract is manipulated, either anatomically or functionally, leading to changed bile acid metabolism. Accumulating evidence has shown that bile acids play a role in metabolic regulation as signaling molecules other than digestive juice. And most of the metabolism-beneficial effects are mediated through nuclear receptor FXR and membrane receptor TGR5, as well as reciprocal influence on gut microbiota. Bile diversion procedure is also performed on animals to recapitulate the benefits of bariatric surgery. It appears that bile acid alteration is an important component of bariatric surgery, and represents a promising target for the management of metabolic disorders