A multi-herb-combined remedy to overcome hyper-inflammatory response by reprogramming transcription factor profile and shaping monocyte subsets.

Traditional Chinese multi-herb-combined prescriptions usually show better performance than a single agent since a group of effective compounds interfere multiple disease-relevant targets simultaneously. Huang-Lian-Jie-Du decoction is a remedy made of four herbs that are widely used to treat oral ulcers, gingivitis, and periodontitis. However, the active ingredients and underlying mechanisms are not clear. To address these questions, we prepared a water extract solution of Huang-Lian-Jie-Du decoction (HLJDD), called it as WEH (Water Extract Solution of HLJDD), and used it to treat LPS-induced systemic inflammation in mice. We observed that WEH attenuated inflammatory responses including reducing production of cytokines, chemokines and interferons (IFNs), further attenuating emergency myelopoiesis, and preventing mice septic lethality. Upon LPS stimulation, mice pretreated with WEH increased circulating Ly6C- patrolling and splenic Ly6C+ inflammatory monocytes. The acute myelopoiesis related transcriptional factor profile was rearranged by WEH. Mechanistically we confirmed that WEH interrupted LPS/TLR4/CD14 signaling-mediated downstream signaling pathways through its nine principal ingredients, which blocked LPS stimulated divergent signaling cascades, such as activation of NF-κB, p38 MAPK, and ERK1/2. We conclude that the old remedy blunts LPS-induced "danger" signal recognition and transduction process at multiple sites. To translate our findings into clinical applications, we refined the crude extract into a pure multicomponent drug by directly mixing these nine chemical entities, which completely reproduced the effect of protecting mice from lethal septic shock. Finally, we reduced a large number of compounds within a multi-herb water extract to seven-chemical combination that exhibited superior therapeutic efficacy compared with WEH.

Recent advances and applications of molecularly imprinted polymers in solid-phase extraction for real sample analysis.

Sample pretreatment is essential for the analysis of complicated real samples due to their complex matrices and low analyte concentrations. Among all sample pretreatment methods, solid-phase extraction is arguably the most frequently used one. However, the majority of available solid-phase extraction adsorbents suffer from limited selectivity. Molecularly imprinted polymers are a type of tailor-made artificial antibodies and receptors with specific recognition sites for target molecules. Using molecularly imprinted polymers instead of conventional adsorbents can greatly improve the selectivity of solid-phase extraction, and therefore molecularly imprinted polymer-based solid-phase extraction has been widely applied to separation, clean up and/or preconcentration of target analytes in various kinds of real samples. In this article, after a brief introduction, the recent developments and applications of molecularly imprinted polymer-based solid-phase extraction for determination of different analytes in complicated real samples during the 2015-2020 are reviewed systematically, including the solid-phase extraction modes, molecularly imprinted adsorbent types and their preparations, and the practical applications of solid-phase extraction to various real samples (environmental, food, biological, and pharmaceutical samples). Finally, the challenges and opportunities of using molecularly imprinted polymer-based solid-phase extraction for real sample analysis are discussed.

Coupling sodium dodecyl sulfate-capillary polyacrylamide gel electrophoresis with matrix-assisted laser desorption ionization time-of-flight mass spectrometry via a poly(tetrafluoroethylene) membrane.

Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) is a fundamental analytical technique for proteomic research, and SDS-capillary gel electrophoresis (CGE) is its miniaturized version. Compared to conventional slab-gel electrophoresis, SDS-CGE has many advantages such as increased separation efficiency, reduced separation time, and automated operation. SDS-CGE is not widely accepted in proteomic research primarily due to the difficulties in identifying the well-resolved proteins. MALDI-TOF-MS is an outstanding platform for protein identifications. Coupling the two would solve the problem but is extremely challenging because the MS detector has no access to the SDS-CGE-resolved proteins and the SDS interferes with MS detection. In this work we introduce an approach to address these issues. We discover that poly(tetrafluoroethylene) (PTFE) membranes are excellent materials for collecting SDS-CGE-separated proteins. We demonstrate that we can wash off the SDS bound to the collected proteins and identify these proteins on-membrane with MALDI-TOF-MS. We also show that we can immunoblot and Coomassie-stain the proteins collected on these membranes.

Flow batteries for microfluidic networks: configuring an electroosmotic pump for nonterminal positions.

A micropump provides flow and pressure for a lab-on-chip device, just as a battery supplies current and voltage for an electronic system. Numerous micropumps have been developed, but none is as versatile as a battery. One cannot easily insert a micropump into a nonterminal position of a fluidic line without affecting the rest of the fluidic system, and one cannot simply connect several micropumps in series to enhance the pressure output, etc. In this work we develop a flow battery (or pressure power supply) to address this issue. A flow battery consists of a +EOP (in which the liquid flows in the same direction as the field gradient) and a -EOP (in which the liquid flows opposite to the electric field gradient), and the outlet of the +EOP is directly connected to the inlet of the -EOP. An external high voltage is applied to this outlet-inlet joint via a short gel-filled capillary that allows ions but not bulk liquid flow, while the +EOP's inlet and the -EOP's outlet (the flow battery's inlet and outlet) are grounded. This flow battery can be deployed anywhere in a fluidic network without electrically affecting the rest of the system. Several flow batteries can be connected in series to enhance the pressure output to drive HPLC separations. In a fluidic system powered by flow batteries, a hydraulic equivalent of Ohm's law can be applied to analyze system pressures and flow rates.

Nanocapillaries for open tubular chromatographic separations of proteins in femtoliter to picoliter samples.

We have recently examined the potential of bare nanocapillaries for free solution DNA separations and demonstrated efficiencies exceeding 10(6) theoretical plates/m. In the present work, we demonstrate the use of bare and hydroxypropylcellulose (HPC) coated open tubular nanocapillaries for protein separations. Using 1.5 microm inner diameter (i.d.) capillary columns, hydrodynamically injecting femto- to picoliter volumes of fluorescent or fluorescent dye labeled protein samples, utilizing a pneumatically pressurized chamber containing 1.0 mM sodium tetraborate solution eluent (typically 200 psi) as the pump, and performing on-column detection using a simple laser-induced fluorescence detector, we demonstrate efficiencies of close to a million theoretical plates/m while generating single digit microliter volumes of waste for a complete chromatographic run. We achieve baseline resolution for a protein mixture consisting of transferrin, alpha-lactalbumin, insulin, and alpha-2-macroglobulin.

Novel microporous ß-cyclodextrin polymer as sorbent for solid-phase extraction of bisphenols in water samples and orange juice.

A new microporous ß-cyclodextrin polymer (MP-CDP) was prepared for the simultaneous solid-phase extraction (SPE) of bisphenol F (BPF), bisphenol A (BPA) and bisphenol AF (BPAF). The MP-CDP was characterized by Fourier transform infrared spectroscopy, solid-phase 13C nuclear magnetic resonance, thermo-gravimetric analysis, scanning electron microscopy, and nitrogen adsorption and desorption analysis. Results indicated that the MP-CDP had micron-level particle size, microporous structure, a high BET surface area, and a high product yield. Adsorption tests showed that the MP-CDP sorbent had high adsorption ability and fast binding kinetics for bisphenols. Moreover, the MP-CDP presented high extraction efficiencies, high enrichment factor, good reusability and good batch-to-batch reproducibility for SPE of bisphenols. Based on the MP-CDP sorbent, a SPE-HPLC-UV method was developed and successfully applied to simultaneously detect three bisphenols in water samples and orange juice with the recoveries of 95.7-106.3% (RSD = 2.0-5.2%) for BPF, 92.9-107.0% (RSD = 1.5-5.1%) for BPA, and 96.0-103.5% (RSD = 1.7-5.0%) for BPAF, respectively. The limit of detection (S/N = 3) and the limit of quantification (S/N = 10) for all bisphenols in these samples were 0.15 ng/mL and 0.5 ng/mL, respectively. The new MP-CDP can be potentially utilized as a good sorbent for simultaneous SPE of trace bisphenols in environmental water samples and beverages.

Two-dimensional liquid chromatography consisting of twelve second-dimension columns for comprehensive analysis of intact proteins.

A comprehensive two-dimensional liquid chromatography (LCxLC) system consisting of twelve columns in the second dimension was developed for comprehensive analysis of intact proteins in complex biological samples. The system consisted of an ion-exchange column in the first dimension and the twelve reverse-phase columns in the second dimension; all thirteen columns were monolithic and prepared inside 250 µm i.d. capillaries. These columns were assembled together through the use of three valves and an innovative configuration. The effluent from the first dimension was continuously fractionated and sequentially transferred into the twelve second-dimension columns, while the second-dimension separations were carried out in a series of batches (six columns per batch). This LCxLC system was tested first using standard proteins followed by real-world samples from E. coli. Baseline separation was observed for eleven standard proteins and hundreds of peaks were observed for the real-world sample analysis. Two-dimensional liquid chromatography, often considered as an effective tool for mapping proteins, is seen as laborious and time-consuming when configured offline. Our online LCxLC system with increased second-dimension columns promises to provide a solution to overcome these hindrances.

Controlled growth and differentiation of MSCs on grooved films assembled from monodisperse biological nanofibers with genetically tunable surface chemistries.

The search for a cell-supporting scaffold with controlled topography and surface chemistry is a constant topic within tissue engineering. Here we have employed M13 phages, which are genetically modifiable biological nanofibers (∼ 880 nm long and ∼ 6.6 nm wide) non-toxic to human beings, to form films for supporting the growth of mesencymal stem cells (MSCs). Films were built from nearly parallel phage bundles separated by grooves. The bundles can guide the elongation and alignment of MSCs along themselves. Phage with peptides displayed on the surface exhibited different control over the fine morphologies and differentiation of the MSCs. When an osteogenic peptide was displayed on the surface of phage, the proliferation and differentiation of MSCs into osteoblasts were significantly accelerated. The use of the grooved phage films allows us to control the proliferation and differentiation of MSCs by simply controlling the concentrations of phages as well as the peptides displayed on the surface of the phages. This work will advance our understanding on the interaction between stem cells and proteins.

A robust cross-linked polyacrylamide coating for microchip electrophoresis of dsDNA fragments.

Surface derivatization plays an important role in microchip electrophoresis. It not only enhances the resolution, but also improves the reproducibility. So far, the most popularly used derivatization method for glass microchannels is to covalently attach a layer of linear polyacrylamide (LPA) to the channel surfaces. However, LPA coating has two problems: incomplete coverage and limited lifetime. To address these issues, we have recently developed a cross-linked polyacrylamide (CPA) derivatization protocol and demonstrated it for high-resolution protein separations by CIEF, CGE, and CZE. In this report, we used this protocol to coat microchip channels and exhibited the reliability and robustness of CPA coating for microchip electrophoresis of DNA molecules. dsDNA fragments were used as our test samples. High resolutions were obtained for fragments ranging from 100 bp to 10 kpb. After more than 800 runs, the CPA-coated microchannels still performed well and comparable resolutions were maintained throughout these runs.

Comprehensive protein profiling by multiplexed capillary zone electrophoresis using cross-linked polyacrylamide coated capillaries.

We have recently developed a new process to create cross-linked polyacrylamide (CPA) coatings on capillary walls to suppress protein-wall interactions. Here, we demonstrate CPA-coated capillaries for high-efficiency (>2 x 10(6) plates per meter) protein separations by capillary zone electrophoresis (CZE). Because CPA virtually eliminates electroosmotic flow, positive and negative proteins cannot be analyzed in a single run. A "one-sample-two-separation" approach is developed to achieve a comprehensive protein analysis. High throughput is achieved through a multiplexed CZE system.

Replaceable cross-linked polyacrylamide for high performance separation of proteins.

An alternative sieving matrix, replaceable cross-linked polyacrylamide (rCPA), was developed for sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) separation of proteins. This rCPA could be conveniently pressurized into separation capillaries under a pressure of 80 psi. SDS-CGE separations using this matrix generated high resolutions for a wide range (approximately 4 kD to approximately 300 kD) of proteins. When compared to the most frequently used sieving matrixes, the rCPA permitted the highest resolutions with comparable or increased separation speed for protein separations.

Cholesterol modulated antibody binding in supported lipid membranes as determined by total internal reflectance microscopy on a microfabricated high-throughput glass chip.

A high-throughput microfabricated all-glass microchip, lipid biochip, was created and used to measure fluorescently tagged antibody binding to dinitrophenol (DNP) haptens in planar supported phospholipid/cholesterol lipid bilayers as a function of cholesterol-to-lipid molar ratio (X(CHOL)). Multiple parallel microchannels etched in the lipid biochip allowed simultaneous measurement of antibody binding to hapten-containing and hapten-free lipid bilayers, for a range of aqueous antibody concentrations. Specific and nonspecific antibody binding to the supported lipid bilayers was determined from the internally calibrated intensity of the surface fluorescence using total internal reflectance fluorescence (TIRF) microscopy. The TIRF intensity data of the specific antibody binding were fitted to the Langmuir isotherm and Hill equation models to determine the apparent dissociation constant K(d), the maximum fluorescence parameter F(infinity), and binding cooperativity n. As X(CHOL) increased from 0 to 0.50, K(d) exhibited a minimum of approximately 4 microM and n reached a maximum of approximately 2.2 at X(CHOL) approximately 0.20. However, F(infinity) appeared to be insensitive to the cholesterol content. The nonspecific binding fraction (NS), defined as the ratio of the TIRF intensity for hapten-free bilayers to that with hapten, showed a minimum of approximately 0.08 also at X(CHOL) approximately 0.20. The results suggest that cholesterol regulates the specific binding affinity and cooperativity, as well as suppresses nonspecific binding of aqueous antibody to a planar supported lipid bilayer surface at an optimal cholesterol content of X(CHOL) approximately 0.20. Interestingly, for X(CHOL) approximately 0.40, NS reached a maximum of approximately 0.57, suggesting significant packing defects in the lipid bilayer surface, possibly as a result of lipid domain formation as predicted by the lipid superlattice model. We conclude that cholesterol plays a significant role in regulating both specific and nonspecific antibody/antigen binding events on the lipid bilayer surface and that our lipid biochip represents a new and useful high-resolution microfluidic device for measuring lipid/protein surface binding activities in a parallel and high-throughput fashion.

Cross-linked polyacrylamide coating for capillary isoelectric focusing.

Polyacrylamide has been used for capillary wall coating for decades. The coating chemistry includes two main steps: (i) attachment of a bifunctional reagent containing a vinyl group to the silica surface and (ii) extension of the anchored vinyl groups through acrylamide polymerization. Since the introduction of this method, many modifications and improvements have been made. However, few of them are successful for routine capillary isoelectric focusing. One of the major problems is the presence of some microspots in which the silica surfaces are poorly coated. Cross-linking the polyacrylamide molecules anchored around these poorly coated spots seems to be a straightforward solution to this problem. Attempts have been made toward this direction, but cross-linked polyacrylamide coatings have not been demonstrated to be much superior over linear polyacrylamide ones. In this report, we have reexamined this approach and demonstrated that cross-linked polyacrylamide could be excellent for capillary isoelectric focusing. A simple device and a new coating protocol have been developed to produce this coating reliably and reproducibly. Compared to the commercial linear polyacrylamide and hydroxypropyl cellulose coatings for CIEF, the cross-linked polyacrylamide coating is much more stable and robust although their initial performances are comparable.