Wash Buffer Research Articles

Generic detection of organophosphorus nerve agent adducts to butyrylcholinesterase in plasma using liquid chromatography-tandem mass spectrometry combined with an improved procainamide-gel separation and pepsin digestion method

Organophosphorus nerve agent (OPNA) adducts to butyrylcholinesterase (BChE) can be applied to confirm exposure in humans. A sensitive method for generic detection of G- and V-series OPNA adducts to BChE in plasma was developed by combining an improved procainamide-gel separation (PGS) and pepsin digestion protocol with ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Residual matrix interferences from prior PGS purification of OPNA-BChE adducts from plasma were found to be a critical cause of significantly reduced UHPLC-MS/MS detection sensitivity. In our developed on-column PGS approach, the matrix interference was successfully removed by adding an appropriate concentration of NaCl to the washing buffer, and it could capture ≥92.5% of the BChE in plasma. The lower pH value and the longer digestion time in all previous pepsin digestion methods were found to be a key accelerated aging factor of several adducts such as tabun (GA)-, cyclohexylsarin (GF)-, and soman (GD)-BChE nonapeptide adducts, making them difficult to detect. The aging event of several OPNA-BChE nonapeptide adducts was so successfully addressed that the formic acid level in enzymatic buffer and digestion time were lowered to 0.05% (pH 2.67) and 0.5 h, respectively, and the post-digestion reaction was immediately terminated. The improved condition parameters were optimal for pepsin digestion of all types of OPNA-BChE adducts into their individual unaged nonapeptide adducts with the highest yields, expanding the applicability of the method. The method had a nearly one-fold decrease in sample preparation time through the reduction of digestion time and removal of ultrafiltration procedure after digestion. The limit of identification (LOI) were determined respectively as 0.13 ng mL−1, 0.28 ng mL−1, 0.50 ng mL−1, 0.41 ng mL−1 and 0.91 ng mL−1 for VX-, sarin (GB)-, GA-, GF-, and GD-exposed human plasma, being low exposure value compared to previously documented approaches. The approach was utilized to fully characterize the adducted (aged and unaged) BChE levels of five OPNAs in a series of their individual exposed concentration (1.00–400 nM) of plasma sample, and successfully detect OPNA exposure from all unknown plasma samples from OPCW's second and third biomedical proficiency tests. The OPNA-BChE adducts, their aged adducts, and unadducted BChE from OPNA-exposed plasma can simultaneously be measured using the method. The study provides a recommended diagnostic tool for generic verification of any OPNA exposure with high confidence by detecting its corresponding BChE adduct.

Separation and Washing of Candida Cells from White Blood Cells Using Viscoelastic Microfluidics

An early and accurate diagnosis of Candida albicans is critical for the rapid antifungal treatment of candidemia, a mortal bloodstream infection. This study demonstrates viscoelastic microfluidic techniques for continuous separation, concentration, and subsequent washing of Candida cells in the blood. The total sample preparation system contains two-step microfluidic devices: a closed-loop separation and concentration device and a co-flow cell-washing device. To determine the flow conditions of the closed-loop device, such as the flow rate factor, a mixture of 4 and 13 μm particles was used. Candida cells were successfully separated from the white blood cells (WBCs) and concentrated by 74.6-fold in the sample reservoir of the closed-loop system at 800 μL/min with a flow rate factor of 3.3. In addition, the collected Candida cells were washed with washing buffer (deionized water) in the microchannels with an aspect ratio of 2 at a total flow rate of 100 μL/min. Finally, Candida cells at extremely low concentrations (Ct > 35) became detectable after the removal of WBCs, the additional buffer solution in the closed-loop system (Ct = 30.3 ± 1.3), and further removal of blood lysate and washing (Ct = 23.3 ± 1.6).

Dissolution-Enhanced Luminescence Enhanced Digital Microfluidics Immunoassay for Sensitive and Automated Detection of H5N1.

Protein bioassay is a critical tool for the screening and detection of protein biomarkers in disease diagnostics and biological applications. However, the detection sensitivity and system automation of current immunoassays do not meet the emerging demands of clinical applications. Here, we developed a dissolution-enhanced luminescence-enhanced digital microfluidics immunoassay (DEL-DMF), which significantly improves the sensitivity and automation of the protein bioassay. In DEL-DMF, the sample and reagent droplets are controlled to complete the processes of sample transport, immunoreaction, and buffer washing, which not only minimizes sample consumption to 2 μL and enhances the binding efficiency of immunoreaction but also streamlines all the procedures and simplifies the process of immunoassay. Moreover, dissolution-enhanced luminescence using NaEuF4 NPs as nanoprobes boosts the fluorescence and increases the sensitivity of the bioassay. We demonstrate the enhanced analytical performance of our DEL-DMF immunoassay to detect H5N1 hemagglutinin in human serum and saliva. A limit of detection of 1.16 pM was achieved in less than 0.5 h with only 2 μL sample consumption. Overall, our DEL-DMF immunoassay combines the merits of the microfluidics platform and dissolution-enhanced luminescence, thus affording superior detection sensitivity and system automation for protein biomarkers. This novel immunoassay microsystem holds great potential in clinical and biological applications.

Investigating thrombin-loaded fibrin in whole blood clot microfluidic assay via fluorogenic peptide

During clotting under flow, thrombin rapidly generates fibrin, whereas fibrin potently sequesters thrombin. This co-regulation was studied using microfluidic whole blood clotting on collagen/tissue factor, followed by buffer wash, and a start/stop cycling flow assay using the thrombin fluorogenic substrate, Boc-Val-Pro-Arg-AMC. After 3 min of clotting (100 s−1) and 5 min of buffer wash, non-elutable thrombin activity was easily detected during cycles of flow cessation. Non-elutable thrombin was similarly detected in plasma clots or arterial whole blood clots (1000 s−1). This thrombin activity was ablated by Phe-Pro-Arg-chloromethylketone (PPACK), apixaban, or Gly-Pro-Arg-Pro to inhibit fibrin. Reaction-diffusion simulations predicted 108 nM thrombin within the clot. Heparin addition to the start/stop assay had little effect on fibrin-bound thrombin, whereas addition of heparin-antithrombin (AT) required over 6 min to inhibit the thrombin, indicating a substantial diffusion limitation. In contrast, heparin-AT rapidly inhibited thrombin within microfluidic plasma clots, indicating marked differences in fibrin structure and functionality between plasma clots and whole blood clots. Addition of GPVI-Fab to blood before venous or arterial clotting (200 or 1000 s−1) markedly reduced fibrin-bound thrombin, whereas GPVI-Fab addition after 90 s of clotting had no effect. Perfusion of AF647-fibrinogen over washed fluorescein isothiocyanate (FITC)-fibrin clots resulted in an intense red layer around, but not within, the original FITC-fibrin. Similarly, introduction of plasma/AF647-fibrinogen generated substantial red fibrin masses that did not penetrate the original green clots, demonstrating that fibrin cannot be re-clotted with fibrinogen. Overall, thrombin within fibrin is non-elutable, easily accessed by peptides, slowly accessed by average-sized proteins (heparin/AT), and not accessible to fresh fibrinogen.

Process Development of Recombinant Adeno-Associated Virus Production Platform Results in High Production Yield and Purity.

Optimization of recombinant adeno-associated virus (rAAV) production has important clinical implications, as manufacturing is one of the major challenges for rAAV gene therapy. In this study, we optimized upstream and downstream processing of the rAAV production platform created by an earlier design-of-experiment approach. Our results showed that adding peptones (yeastolate, Trypton N1 or both) increased production yield by 2.8- to 3.4-folds. For downstream processing, a variety of wash buffers for an affinity resin, POROS™ CaptureSelect™ (PCS)-AAVX, were tested for their effects on rAAV8 purity, including NaCl, MgCl2, arginine, Triton X-100, CHAPS, Tween 20, octyl β-d-1-thioglucopyranoside (OTG), and low pH. The results showed that the OTG wash significantly improved the rAAV purity to 97% and reduced endotoxins to an undetectable level (<0.5 EU/mL), while retaining the yield at 92.3% of the phosphate-buffered saline (PBS) wash. The OTG wash was successfully applied to purifications of rAAV1, rAAV2, and rAAV5 using PCS-AAVX, and rAAV9 using PCS-AAV9. rAAV8 purified with OTG wash showed comparable transduction efficiency in HEK 293T cells to the rAAV8 purified with PBS wash. The optimized rAAV production process yielded 5.5-6.0 × 1014 and 7.6 × 1014 vector genome per liter of HEK 293T cells for purified rAAV8- and rAAV5-EF1α-EGFP (enhanced green fluorescent protein), respectively. The platform described in this study is simple with high yields and purity, which will be beneficial to both research and clinical gene therapy.

Direct capture and amplification of nucleic acids using a universal, elution-free magnetic bead-based method for rapid pathogen detection in multiple types of biological samples.

Nucleic acid amplification tests (NAATs) have become an attractive approach for pathogen detection, and obtaining high-quality nucleic acid extracts from biological samples plays a critical role in ensuring accurate NAATs. In this work, we established an elution-free magnetic bead (MB)-based method by introducing polyethylene-polypropylene glycol (PEPPG) F68 in lysis buffer and using NaOH solution instead of alcohols as the washing buffer for rapid nucleic acid extraction from multiple types of biological samples, including nasopharyngeal swabs, serum, milk, and pork, which bypassed the nucleic acid elution step and allowed the nucleic acid/MB composite to be directly used as the template for amplification reactions. The entire extraction process was able to be completed in approximately 7 min. Even though the nucleic acid/MB composite could not be used for quantitative real-time PCR (qPCR) assays, this elution-free MB-based method significantly improved the sensitivity of the loop-mediated isothermal amplification (LAMP) assay. The sensitivity of the quantitative real-timeLAMP(qLAMP) assays combined with this elution-free MB-based method showed an improvement of one to three orders of magnitude compared with qLAMP or qPCR assays combined with the traditional MB-based method. In addition to manual operation, like the traditional MB-based method, this universal, rapid, and facile nucleic acid extraction method also has potential for integration into automated robotic processing, making it particularly suitable for the establishment of an analysis platform for ultrafast and sensitive pathogen detection in various biological samples both in centralized laboratories and at remote sites.

Effect of solution condition on the binding behaviors of monoclonal antibody and fusion protein therapeutics in Protein A chromatography

Recent development in Protein A chromatography has adopted wash buffers with a wide range of pH to improve the removal of process-related impurities, e.g., host cell proteins (HCPs), from Fc-based target biological products. Thus, it is important to investigate the pH effect on the binding behaviors of target products of various properties on Protein A resins. Here we performed column breakthrough experiments for two monoclonal antibodies (mAbs) and one Fc-fusion protein on two Protein A resins from pH 4 to pH 10, and the experimental data was analyzed using a mechanistic model to obtain isotherm and mass transfer parameters. The two mAbs exhibited relatively high dynamic binding capacity (DBC) at 10% breakthrough of 43 – 67 g/L at pH 6–9 followed by a ∼30% decrease from pH 9 to pH 10; while the Fc-fusion protein reached the highest DBC at 10% breakthrough of 10 – 17 g/L at pH 5 and thereafter the capacity gradually reduced. Model analyses revealed that the two mAbs had higher maximum binding capacity by 2 – 5 fold but weaker binding affinity (12 – 64%) than the Fc-fusion protein from pH 5 to pH 9. For the three molecules, similar patterns of the pH impact were observed on the two Protein A resins with the Jetted A50 resin showing generally higher DBC and stronger binding affinity than the MabSelect SuRe LX resin. Additionally, an inverse relationship between the binding affinity and surface diffusivity was observed for both resins. Besides obtaining the isotherm parameters from the column breakthrough data, a direct batch equilibrium measurement showed comparable trend in these parameters with relatively more scattered values due to the inherent uncertainties to accurately determine the initial slope of the isotherm in highly favorable adsorption conditions. Finally, isothermal titration calorimetry (ITC) results revealed that the measured binding affinity using free Protein A ligand was stronger than that obtained from the breakthrough and isotherm results for the resin, possibly due to the reduced accessibility of the immobilized ligand on resin surface. Overall, this work can facilitate future Protein A ligand design and affinity chromatography process optimization for biomanufacturing.

Optimization for Simultaneous Removal of Product/Process-Related Impurities of Peptide Fc-Fusion Protein Using Cation Exchange Chromatography

Fc fusion proteins are used as therapeutic agents with unique structures by combining the Fc domain of an antibody with other active proteins, cytokines, and enzymes. Peptide Fc-fusion proteins are complex fusion molecules that possess a structure different from that of monoclonal antibodies (mAbs) and are difficult to express, thereby affecting their quality. Many product/process-related impurities generated during the production of peptide Fc-fusion proteins pose a risk to the robustness of pre-existing three-column platforms for the purification of mAbs. Thus, we first evaluated the effect of pH, conductivity, and dynamic binding capacity (DBC; g of product per liter of resin) on the separation of host cell protein (HCP) and high molecular weight (HMW) and low molecular weight (LMW) proteins in strong cation exchange chromatography and then established an operating range using the design of experiments (DoE). Based on our studies, the optimal removal rates of HCP and HMW were achieved under the following conditions: 8 CV of wash buffer, 20–23 g/L of resin DBC, and an elution buffer conductivity of 63–66 mS/cm. The conductivity of the wash buffer used to remove the LMW was 50 mS/cm. In addition, reproducibility was confirmed by scaling up two batches using the Fractogel® EMD SO3− (M) resin. As a result of confirming with a validated test method in all batches, &gt;55% yield, &gt;98.2% purity, and &gt;27% HCP reduction rate were satisfied. The cation exchanger exhibited an acceptable step yield and effectively reduced product/process-related impurities within the established range.

Aptamer-linked photonic crystal hydrogel sensor for rapid point-of-care detection of human immuno-deficiency virus-1 (HIV-1)

The detection of the human immunodeficiency virus-1 (HIV) at an early stage is vital and could be realized through its cell surface glycoprotein-120 (gp120) without virus preprocessing. Here, we present an ssDNA-aptamer-linked photonic crystal (APC) hydrogel sensor for HIV detection which is comprised of photonic crystals (PCs) made of polystyrene nanoparticles embedded in the polyacrylamide hydrogel. ssDNA aptamers specific for gp120 are crosslinked in the hydrogel which can selectively bind to gp120 by hydrogen bonding increasing the PCs particle spacing and swelling of the hydrogel. The binding response can be visually monitored as a color change due to the diffraction of light from PCs and can eventually be measured (1–1000 ng mL−1 of gp120) and 100 to 108 VP mL−1 of HIV by the Debye’s ring diameter or a UV/Vis spectrometer. APC-hydrogel can be regenerated by Tris-HCl and EDTA washing buffer system. The sensor demonstrates LOD of 7.1 ± 1.55 ng mL−1 for gp120 and 4 VP mL−1 for the whole HIV, a rapid response of 5 min, reusability up to 70 % (in fifth use), and recovery of 95.4 ± 0.1 % to 99.0 ± 0.2 % in plasma samples. The sensor is cost-effect and stable compared to antibody-based sensors and can be utilized to develop point-of-care testing (POCT) devices for HIV diagnosis.

Direct amplification of Bordetella pertussis DNA purified from nasopharyngeal swabs by a low-cost, fast (60-second), and equipment-free method.

To develop and evaluate a low-cost cellulose-based method for rapid purification and direct amplification of Bordetella pertussis DNA from nasopharyngeal swabs. We prepared cellulose discs and evaluated different parameters (lysis/wash buffers, number of discs and DNA elution). The method was coupled to a direct real-time PCR (qPCR) amplification and the performance was estimated using nasopharyngeal swabs that were positive (n=100) and negative (n=50) for B. pertussis DNA by qPCR, compared to the silica column-based method. We calculated sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) and the degree of agreement. The feasibility of the rapid method to be coupled to a loop-mediated isothermal amplification colorimetric assay (LAMP) was evaluated. The rapid method, with a cellulose disk and lysis and wash buffer containing PVP-40 and Tween 20, respectively, showed a greater capacity to purify amplifiable DNA from B. pertussis. The method had a sensitivity of 89.0% (95%CI: 80.2%-94.9%) and a specificity of 98.5% (95%CI: 92.1%-100.0%), with a good degree of agreement (Kappa=0.867; 95%CI: 0.788 - 0.946), compared to the reference method. The PPV and NPV were 98.6% (95%CI: 92.7.2%-100.0%) and 88.2% (95%CI: 78.7%-94.4%), respectively. Successful amplification by LAMP was evident, and comparable results were obtained with the silica column method. The developed method is simple, low-cost and equipment-free for rapid (60 seconds) DNA collection at the point of care, and can be implemented in various molecular techniques aimed at the timely diagnosis and epidemiological study of pertussis.

A Flow Cytometry-based Cell Surface Protein Binding Assay for Assessing Selectivity and Specificity of an Anticancer Aptamer.

A key challenge in developing an anticancer aptamer is to efficiently determine the selectivity and specificity of the developed aptamer to the target protein. Due to its several advantages over monoclonal antibodies, aptamer development has gained enormous popularity among cancer researchers. Systematic evolution of ligands by exponential enrichment (SELEX) is the most common method of developing aptamers specific for proteins of interest. Following SELEX, a quick and efficient binding assay accelerates the process of identification, confirming the selectivity and specificity of the aptamer. This paper explains a step-by-step flow cytometric-based binding assay of an aptamer specific for epithelial cellular adhesion molecule (EpCAM). The transmembrane glycoprotein EpCAM is overexpressed in most carcinomas and plays roles in cancer initiation, progression, and metastasis. Therefore, it is a valuable candidate for targeted drug delivery to tumors. To evaluate the selectivity and specificity of the aptamer to the membrane-bound EpCAM, EpCAM-positive and -negative cells are required. Additionally, a non-binding EpCAM aptamer with a similar length and 2-dimensional (2D) structure to the EpCAM-binding aptamer is required. The binding assay includes different buffers (blocking buffer, wash buffer, incubation buffer, and FACS buffer) and incubation steps. The aptamer is incubated with the cell lines. Following the incubation and washing steps, the cells will be evaluated using a sensitive flow cytometry assay. Analysis of the results shows the binding of the EpCAM-specific aptamer to EpCAM-positive cells and not the EpCAM-negative cells. In EpCAM-positive cells, this is depicted as a band shift in the binding of the EpCAM aptamer to the right compared to the non-binding aptamer control. In EpCAM-negative cells, the corresponding bands of EpCAM-binding and -non-binding aptamers overlap. This demonstrates the selectivity and specificity of the EpCAM aptamer. While this protocol is focused on the EpCAM aptamer, the protocol is applicable to other published aptamers.

Culture-Free Quantification of Bacteria Using Digital Fluorescence Imaging in a Tunable Magnetic Capturing Cartridge for Onsite Food Testing.

Accurate, onsite detection of pathogenic bacteria from food matrices is required to rapidly respond to pathogen outbreaks. However, accurately detecting whole-cell bacteria in large sample volumes without an enrichment step remains a challenge. Therefore, bacterial samples must be concentrated, identified, and quantified. We developed a tunable magnetic capturing cartridge (TMCC) and combined it with a portable digital fluorescence reader for quick, onsite, quantitative detection of Staphylococcus aureus. The TMCC platform integrates an absorption pad impregnated with water-soluble polyvinyl alcohol (PVA) with an injection-molded polycarbonate (PC) plate that has a hard magnet on its back and an acrylonitrile-butadiene-styrene case. An S. aureus-specific antibody conjugated with magnetic nanoparticles was used to concentrate bacteria from a large-volume sample and capture bacteria within the TMCC. The retention time for capturing bacteria on the TMCC was adjusted by controlling the concentration and volume of the PVA solution. Concentrated bacterial samples bound to target-specific aptamer probes conjugated with quantum dots were loaded into the TMCC for a controlled time, followed by attachment of the bacteria to the PC plate and removal of unbound aptamer probes with wash buffer. The captured bacteria were quantified using a digital fluorescence reader equipped with an embedded program that automatically counts fluorescently tagged bacteria. The bacterial count made using the TMCC was comparable to a standard plate count (R2 = 0.9898), with assay sensitivity and specificity of 94.3 and 100%, respectively.

Systematic Optimisation of Microtiter Plate Lectin Assay to Improve Sialic Acid Linkage Detection.

We aimed to develop a high-throughput lectin assay with minimized background signals to investigate the interactions of lectins and sialic acid glycans, focusing on Prostate- Specific Antigen (PSA). High background signals resulting from nonspecific binding are a significant concern for microtiter plate-based Enzyme-Linked Lectin Sorbent Assays (ELISAs), as they can mask specific binding signals and cause false-positive results. In this study, we constructed an ELISA based on different washing step parameters, including the number of washing cycles, NaCl and Tween-20 concentrations, and the type of blocking agent and evaluated the effects on both specific and nonspecific binding signals. Furthermore, we performed a PSA binding assay using the optimized ELISA. The optimal washing parameters based on the highest specific binding signal proposed four cycles of washing steps using a washing buffer containing a high salt concentration (0.5 M NaCl) and mild detergent (0.05% Tween-20). The utilization of the optimized washing parameters in this assay was shown to be sufficient to obtain the optimal binding signals without the use of any blocking agent. Binding assays performed using the optimized ELISA revealed that the glycan of the PSA sample used in this study mainly consists of terminal α2,6-linked sialic acid, as strongly recognized by Sambucus nigra agglutinin (SNA) with a KD value of 12.38 nM. The ELISA reported in this study provides a simple yet sensitive assay for sialic acid linkage recognition.

An Integrated, Real-Time Convective PCR System for Isolation, Amplification, and Detection of Nucleic Acids

Convective PCR (CPCR) can perform rapid nucleic acid amplification by inducing thermal convection to continuously, cyclically driving reagent between different zones of the reactor for spatially separate melting, annealing, and extending in a capillary tube with constant heating temperatures at different locations. CPCR is promoted by incorporating an FTA membrane filter into the capillary tube, which constructs a single convective PCR reactor for both sample preparation and amplification. To simplify fluid control in sample preparation, lysed sample or wash buffer is driven through the membrane filter through centrifugation. A movable resistance heater is used to heat the capillary tube for amplification, and meanwhile, a smartphone camera is adopted to monitor in situ fluorescence signal from the reaction. Different from other existing CPCR systems with the described simple, easy-to-use, integrated, real-time microfluidic CPCR system, rapid nucleic acid analysis can be performed from sample to answer. A couple of critical issues, including wash scheme and reaction temperature, are analyzed for optimized system performance. It is demonstrated that influenza A virus with the reasonable concentration down to 1.0 TCID50/mL can be successfully detected by the integrated microfluidic system within 45 min.

An investigation into simplifying total RNA extraction with minimal equipment using a low volume, electrokinetically driven microfluidic protocol.

Current methods for total RNA extraction are time-consuming and require several hands-on steps and specialized equipment. Microfluidic devices can offer the opportunity to reduce the number of hands-on steps, decrease the volumes of reagents required for purification, and make extraction high throughput. Here, we investigated the translation of a high volume magnetic bead-based total RNA extraction method (from human whole blood) onto a low input volume microfluidic device. Our results first show that RNA integrity is maintained when the reagent volumes are scaled down by a factor of 22 and the wash buffers are combined 1:1. With our microfluidic method, the number of wash steps can be reduced from four to one. Thus, the time to complete RNA extraction can be reduced from 2 h to 40 min. These manipulations to the conventional protocol yielded RNA amplifiable within 40 cycles of reverse transcription quantitative PCR (RT-qPCR) when using the microfluidic device to simplify the wash steps. To improve the purification of the RNA during the bead transport through the microchannel, we also investigated the effect of a synergetic application of the electrokinetic flow. Our results show that DNase I and other contaminants surrounding the beads get washed away more effectively via electrophoretic transport. Most notably, RNA adsorption on the beads is strong enough to counter electrophoretically-driven desorption. In all, our work opens new ways to extract high-quality total RNA rapidly and simply from a small quantity of blood, making the process of RNA extraction more accessible.

High Mannose-Specific Aptamers for Broad-Spectrum Virus Inhibition and Cancer Targeting

High Mannose-Specific Aptamers for Broad-Spectrum Virus Inhibition and Cancer Targeting

Cryo-XPS spectra from bacterial reference strain Pseudomonas fluorescens DSM50090

Cryogenic x-ray photoelectron spectroscopy was used to analyze the cell envelope of intact and hydrated Gram-negative bacteria of the species Pseudomonas fluorescens. We used a reference strain, DSM50090, from the German microbial culture collection, which we previously have suggested would function well as a reference strain for future XPS analyses of Gram-negative bacteria. Bacteria were grown on nutrient agar plates at room temperature, collected with a cultivation loop, and washed using phosphate buffered saline. An aliquot of the cell pellet was fast-frozen on the sample holder in the sample introduction chamber to a temperature of 103 K and kept frozen throughout the measurement. Survey spectra and high-resolution spectra of Na 1s, O 1s, N 1s, C 1s, Cl 2p, S 2p, and P 2p are reported. The spectra obtained from the analyzed cells represent a combined signal from O, N, C, and S atoms in proteins, lipids, and polysaccharides at the cell surface. Furthermore, signal from P, Na, K, and Cl atoms was present both originating from processes in the cell envelope and remnants from the wash buffer.

Comparable Results of Helicobacter pylori Antibiotic Resistance Testing of Stools vs Gastric Biopsies Using Next-Generation Sequencing

Comparable Results of Helicobacter pylori Antibiotic Resistance Testing of Stools vs Gastric Biopsies Using Next-Generation Sequencing

Washing Lipoaspirate Improves Fat Graft Survival in Nude Mice

The optimal fat processing technique of fat grafting has not been determined. We have proved the importance of washing lipoaspirate to remove blood, but the necessity of washing when there is no obvious bleeding during liposuction is not clear. The purpose of this study is to further investigate the effect of washing on fat graft survival and the underlying mechanisms, from the perspective of inflammation, oxidative stress and apoptosis. To exclude the influence of blood, de-erythrocyte infranatant (dEI) isolated from lipoaspirate was obtained. Purified fat processed by cotton pad filtration mixed with dEIs after sedimentation (sedimentation group), washing (washing group) or phosphate buffer solution (control group) was transplanted to nude mice subcutaneously. Samples were harvested at 1 day and 1, 3, 8 weeks after transplantation. Volume and weight retention, histologic examination, immunostaining of perilipin-1, CD31, CD45 and Ly6g, mRNA expression of PPAR-γ, C/EBPα, VEGF, bFGF, IL-6, IL10, TNF-α, TGF-β, Bax and Bcl-2, and protein contents of 8-iso-PGF2α, IL-6, IL10, TNF-α and TGF-β were all compared among groups. After transplantation, volume and weight retention, histologic scores, viable adipocytes and vascularization were all improved in the washing group, with increased expression of adipogenic and angiogenic genes. Compared with the sedimentation group, the washing group had milder inflammation, lower levels of oxidative stress and apoptosis. Washing lipoaspirate to eliminate mixed components can improve fat graft survival and promote adipogenesis and angiogenesis, possibly by relieving inflammation, reducing oxidative stress injury and inhibiting apoptosis. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of 47 these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors http://www.springer.com/00266 .

Surface Charge Characteristics of Bacillus Subtilis NRS-762 Cells

Bacterial cell surface carries an electrical charge due to the myriad functional groups present, as well as assortment of ions and molecules non-specifically adsorbed to the cell surface. Thus, solution in contact with the bacterial cell surface play a critical role in influencing the overall surface charge characteristics through conferring non-specifically adsorbed ions and molecules. Various wash buffers are commonly used in removing non-specifically adsorbed ions and molecules for revealing the real surface charge of the bacterium. Using electrophoretic mobility measurement of zeta potential, this study attempted to understand the surface charge characteristics of Bacillus subtilis NRS-762 (ATCC 8473) with the help of three wash buffers: deionized (DI) water, 0.1M sodium nitrate, and 9 g/L sodium chloride. Experiment results revealed that B. subtilis NRS-762 was negatively charged over the entire pH range from 1.5 to 12. Specifically, with deionized water as wash buffer, the point-of-zero-charge (pHzpc) was at pH 1.5, which indicated that large amount of negatively charged functional groups were present on the cell surface. Comparison between the zeta potential-pH profiles of B. subtilis NRS-762 cultivated at 30 oC and 37 oC revealed that the profile for growth at 37 oC was more negatively charged over the entire pH range compared to that for growth at 30 oC. This highlighted that physiological adaptation might had occurred on the cell surface for coping with growth at a higher temperature. Zeta potential-pH profiles obtained revealed that DI water could not remove significant quantities of the non-specifically adsorbed ions and molecules. On the other hand, the zeta potential-pH profiles of cells washed with 0.1M sodium nitrate and 9 g/L sodium chloride overlapped each other substantially and were more negatively charged over the pH range from 2 to 11, compared to that of cells washed with DI water. This revealed substantial removal of non-specifically adsorbed ions and molecules with the use of 0.1M sodium nitrate (0.1M ionic strength) and 9 g/L sodium chloride (0.15M ionic strength), which helped reveal the actual surface charge of B. subtilis NRS-762 cells. Collectively, actual surface charge of B. subtilis NRS-762 was masked by non-specifically adsorbed ions and molecules, which could be removed by 0.1M sodium nitrate and 9 g/L sodium chloride wash buffer. Thus, in the case of B. subtilis NRS-762, 0.1M ionic strength wash buffer was the threshold at which there was complete removal of non-specifically adsorbed ions and molecules from the cell surface.