Dilute Buffer Research Articles

Backbone and side chain 1H, 15N and 13C chemical shift assignments of the molten globule state of L94G mutant of horse cytochrome-c.

Proteins fold via a number of intermediates that help them to attain their unique native 3D structure. These intermediates can be trapped under extreme conditions of pH, temperature and chemical denaturants. Similar states can also be achieved by other processes like chemical modification, site directed mutagenesis (or point mutation) and cleavage of covalent bonds of natural proteins under physiological conditions usually taken as dilute buffer (near neutral pH) and 25°C. Structural characterization of molten globules is hampered due to (i) their transient nature, (ii) very low population at equilibrium, and (iii) prone to aggregation at high concentration. Furthermore, the dynamic nature of these folding intermediates makes them unsuitable for X-ray diffraction. Hence, understanding their structures at the atomic level is often a challenge. However, characterization of these intermediates at the atomic level is possible by NMR, which could possibly unravel new details of the protein folding process. We have previously shown that the L94G mutant of horse cytochrome-c displays characteristics of the molten globule (MG) state at pH 6.0 and 25°C. As a first step towards characterizing this MG state at the atomic level by NMR, we report its complete backbone, side chain and heme chemical shift assignments.

Methanesulfonyl Iodide

Methanesulfonyl iodide is produced in aqueous solutions from the reaction of triiodide with methanesulfinate. Dichroic crystals of (CH3SO2I)4·KI3·2I2 are formed from KI/I2 solutions with high concentrations of CH3SO2-, while dichroic crystals of (CH3SO2I)2·RbI3 are formed from RbI/I2 solutions. X-ray crystallography of these two compounds shows that the CH3SO2I molecules coordinate through their oxygen atoms to the metal cations and that the S-I bond length is 2.44 Å. At low concentrations of CH3SO2-, the solutions remain homogeneous and the sulfonyl iodide is formed in a rapid equilibrium: CH3SO2- + I3- ⇌ CH3SO2I + 2I-, KMSI = 1.07 ± 0.01 M at 25 °C (μ = 0.1 M, NaClO4). The sulfonyl iodide solutions display an absorbance maximum at 309 nm with a molar absorptivity of 667 M-1 cm-1. Stopped-flow studies reveal that the equilibrium is established within the dead time of the instrument (∼2 ms). Solutions of CH3SO2I decompose slowly to form the sulfonate: CH3SO2I + H2O → CH3SO3- + I- + 2H+, khyd. In dilute phosphate buffer, this decomposition occurs with khyd = 2.0 × 10-4 s-1; the decomposition rate shows an inverse-squared dependence on [I-] because of the KMSI equilibrium.

Effects of the routine multiple vaccinations on the expression of innate immune molecules and induction of histone modification in ovarian cells of layer chicks

The aim of this study was to determine whether vaccination affects the expression of Toll-like receptors (TLRs), cytokines, and avian β-defensins (AvBDs) in the chick ovary with or without lipopolysaccharide (LPS) stimulation. White Leghorn female chicks were administered vaccines for infectious bronchitis, Marek's disease, Newcastle disease, and infectious bursal disease during the first 14 D after hatching and ovarian tissues were collected on day 21. Control chicks received water or dilution buffer in place of vaccine. In Experiment 1, ovarian tissues were incubated with or without LPS, and the expression of innate immune molecules (TLRs, cytokines, and AvBDs) was examined by real-time PCR. In Experiment 2, the levels of histone modification in fresh ovarian tissues were examined by western blot analysis. The results of Experiment 1 showed that, in vaccinated chick ovaries, the expression of TLR1-1, 2-1, 2-2, and 21 was up-regulated, whereas that of TLR1-2, 4, and 7 was down-regulated under LPS stimulation. Among the examined 6 cytokines, only the expression of TNFSF15 was lower in the ovaries of vaccinated chicks than that in control with or without LPS stimulation. The expression of AvBD1, 2, 4, and 7 was lower in the ovaries of vaccinated chicks than in control without LPS stimulation, and that of AvBD1 and 2 was also lower even in ovaries incubated with LPS. In Experiment 2, the density of di-methyl histone H3 (Lys9) and acetyl histone H3 (Lys9) was significantly higher in the vaccine group than in the control, whereas di-methyl and tri-methyl histone H3 (Lys4) and acetyl histone H3 (Lys27) did not show differences between the groups. These results suggest that vaccination positively or negatively affects the expression of innate immune molecules in the chick ovary including TLRs, TNFSF15, and AvBDs, and it may be associated with epigenetic reprogramming by histone modifications in ovarian cells. Thus, in the future, it may be possible to develop or improve vaccination programs for the enhancement of the innate immune system in the hen ovary.

Rapid Diagnostic Test Kit for Point-of-Care Cerebrospinal Fluid Leak Detection.

Cerebrospinal fluid (CSF) leaks can occur when there is communication between the intracranial cavities and the external environment. They are a common and serious complication of numerous procedures in otolaryngology, and if not treated, persistent leaks can increase a patient's risk of developing life-threatening complications such as meningitis. As it is not uncommon for patients to exhibit increased secretions postoperatively, distinguishing normal secretions from those containing CSF can be difficult. Currently, there are no proven, available tests that allow a medical provider concerned about a CSF leak to inexpensively, rapidly, and noninvasively rule out the presence of a leak. The gold standard laboratory-based test requires that a sample be sent to a tertiary site for analysis, where days to weeks may pass before results return. To address this, our group recently developed a semiquantitative, barcode-style lateral-flow immunoassay (LFA) for the quantification of the beta-trace protein, which has been reported to be an indicator of the presence of CSF leaks. In the work presented here, we created a rapid diagnostic test kit composed of our LFA, a collection swab, dilution buffers, disposable pipettes, and instructions. Validation studies demonstrated excellent predictive capabilities of this kit in distinguishing between clinical specimens containing CSF and those that did not. Our diagnostic kit for CSF leak detection can be operated by an untrained user, does not require any external equipment, and can be performed in approximately 20 min, making it well suited for use at the point of care. This kit has the potential to transform patient outcomes.

Laser-Assisted Evaporative Cooling of Anions.

We report the first cooling of atomic anions by laser radiation. O^{-} ions confined in a linear Paul trap were cooled by selectively photodetaching the hottest particles. For this purpose, anions with the highest total energy were illuminated with a 532nm laser at their maximal radial excursion. Using laser-particle interaction, we realized a both colder and denser ion cloud, achieving a more than threefold temperature reduction from 1.15 to 0.33eV. Compared with the interaction with a dilute buffer gas, the energy-selective addressing and removal of anions resulted in lower final temperatures, yet acted 10 times faster and preserved twice as large a fraction of ions in the final state. An ensemble of cold negative ions affords the ability to sympathetically cool any other negative ion species, enabling or facilitating a broad range of fundamental studies from interstellar chemistry to antimatter gravity. The technique can be extended to any negative ion species that can be neutralized via photodetachment.

Improved hydrolysis of α-tocopherol acetate emulsion and its bioaccessibility in the presence of polysaccharides and PEG2000

Dietary macromolecules are often used for food formulation. Most of the published literature usually focuses on a certain polysaccharide reducing lipid digestion, but the non-specific physical effects such as the excluded volume effect of the surrounding macromolecules, which will possibly enhance the hydrolysis of lipids, is rarely discussed. As the influence of polysaccharides on the gastrointestinal fate of α-tocopherol acetate (VE-A) encapsulated in emulsion have not been conducted so far, in this research, we study the effect of crowded medium created by polysaccharides (Ficoll400) on the hydrolysis of VE-A and its bioaccessibility. The results show that the presence of Ficoll400 increased the bioaccessibility and conversion of VE-A 1.6 times that of a dilute buffer. An enzyme-catalyzed kinetic reaction rate of VE-A hydrolysis is increased up to 3.5 times. Both the improved thermodynamic conversion yields and kinetic rates of the hydrolysis of the VE-A emulsion was further observed in a synthetic macromolecule PEG2000 medium. This research improves our understanding of the crowding effect of macromolecules on VE-A/lipid emulsion digestion.

Controlling and quantifying protein concentration in Escherichia coli.

The cellular environment is dynamic and complex, involving thousands of different macromolecules with total concentrations of hundreds of grams per liter. However, most biochemistry is conducted in dilute buffer where the concentration of macromolecules is less than 10 g/L. High concentrations of macromolecules affect protein stability, function, and protein complex formation, but to understand these phenomena fully we need to know the concentration of the test protein in cells. Here, we quantify the concentration of an overexpressed recombinant protein, a variant of the B1 domain of protein G, in Tuner (DE3)™ Escherichia coli cells as a function of inducer concentration. We find that the protein expression level is controllable, and expression saturates at over 2 mM upon induction with 0.4 mM isopropyl β-d-thiogalactoside. We discuss the results in terms of what can and cannot be learned from in-cell protein NMR studies in E. coli.

Optimizing the Method of Cell Separation from Bile of Patients with Cholangiocarcinoma for Flow Cytometry.

Analysis of the change of the cells in bile is an evolving research field in biliary pathophysiology and has potential value in diagnosis and therapy. However, laboratory studies of cell in bile across the world are scarce. Bile was collected from the clinical patients with cholangiocarcinoma (CC). To optimize the cell separation method in bile of patients with CC, we studied the factors that may affect cell vitality in bile including the dilution buffer, centrifugal force, centrifugal time, and store time and temperature. Then these factors were modified and performance was evaluated by flow cytometry with respect to the percentage and total yield of viable cells. The separated cells from bile were stained with CD3, CD4, CD8, CD56, TCRγ/δ, CD16, CD14, HLA-DR, CD33, CD15, CD11b, lineage cocktail (CD3, CD14, CD19, CD20, and CD56), CD66b, and CD45 antibodies. The different buffer solutions were joined in bile of patients with CC; experiment results show that the different dilutions have nearly no effect on the ratio of cells in bile by flow cytometry. The best centrifugal procedure was 300 g, 10 min. Bile should be stored at 4°C rather than at normal temperature. Our study further showed that the shorter time of the bile storage, the higher viability of the cell, and immune cells existed in cells isolated from bile. Evaluating bile cell viability is necessary to evaluate method performance.

Macromolecular crowding modulates actin bundle formation induced by actin crosslinking proteins

Bundles of filamentous actin generate high ordered cellular structures including filopodia and lamellipodia with the help of actin binding proteins. α‐actinin and fascin are essential actin crosslinking proteins that affect the mechanical properties of bundles. While the roles of both actin binding proteins in bundling have been well studied in dilute buffer conditions, how they affect bundle formation in crowded environments is not established. Here, we investigate the effects of macromolecular crowding on actin bundles crosslinked by α‐actinin and fascin in vitro and in silico. Time‐dependent bundling processes by both proteins were monitored in the presence of the polymeric crowding agent polyethylene glycol (PEG) using total internal reflection fluorescence (TIRF) microscopy. Biophysical analysis show that PEG reduces the amount and average lengths of α‐actinin‐induced bundles but promotes the formation of thicker bundles induced by fascin. Utilizing molecular dynamics (MD) simulations, we evaluated the interaction between actin filaments and bundling proteins by measuring the contact area and the number of hydrogen bonds. The simulation results demonstrate that PEG increases the binding affinities between actin and binding domain of fascin, while lowering the affinities between actin and α‐actinin. Taken together, our work suggests that crowded environments may modulate actin bundling induced by actin binding proteins in vivo.Support or Funding InformationThis study was supported by the UCF start‐up fund and In‐House grant for Hyeran Kang. We acknowledge the computational time from UCF stokes cluster.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Measurement of anti-influenza neuraminidase antibody induced by H7N9 influenza vaccine

Objective To develop an enzyme-linked lectin assay (ELLA) for measuring neuraminidase inhibition (NI) antibody titers in subjects vaccinated with H7N9 influenza vaccine. Methods Neuraminidase substrate, the dilution and incubation time of enzyme-labeled antibody, the concentration of influenza antigen for coating and pH value of the dilution buffer were optimized. Based on that, ELLA was established and used to detect anti-influenza neuraminidase antibody titers in serum samples of 34 subjects before and after vaccination with H7N9 influenza vaccine. Results The optimal neuraminidase substrate was fetuin at a coating concentration of 7.5 μg/ml. The optimal dilution of enzyme-labeled antibody was 1∶500. The virus strain of influenza H7N9 vaccine was used as antigen at a concentrations of 4.5lgCCID50/ml in solution with a pH of 6.5. Influenza-specific NI titers detected after immunization with vaccine were significantly higher than those before vaccination (P<0.001). In the 34 subjects receiving H7N9 vaccine, the seroconversion rate of NI antibody was 47% (≥40 in NI titer ), which was lower than that of HI antibody (P<0.05). Conclusions An ELLA with natural substrate for measurement of anti-influenza NI antibody was developed. It is simple and practical and might be used in the establishment of immune evaluation system for influenza vaccines and NI antibody. Key words: H7N9 influenza vaccine; Neuraminidase inhibition antibody; ELLA

Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes

BackgroundTransgenic zebrafish lines with the expression of a fluorescent reporter under the control of a cell-type specific promoter, enable transcriptome analysis of FACS sorted cell populations. RNA quality and yield are key determinant factors for accurate expression profiling. Limited cell number and FACS induced cellular stress make RNA isolation of sorted zebrafish cells a delicate process. We aimed to optimize a workflow to extract sufficient amounts of high-quality RNA from a limited number of FACS sorted cells from Tg(fli1a:GFP) zebrafish embryos, which can be used for accurate gene expression analysis.ResultsWe evaluated two suitable RNA isolation kits (the RNAqueous micro and the RNeasy plus micro kit) and determined that sorting cells directly into lysis buffer is a critical step for success. For low cell numbers, this ensures direct cell lysis, protects RNA from degradation and results in a higher RNA quality and yield. We showed that this works well up to 0.5× dilution of the lysis buffer with sorted cells. In our sort settings, this corresponded to 30,000 and 75,000 cells for the RNAqueous micro kit and RNeasy plus micro kit respectively. Sorting more cells dilutes the lysis buffer too much and requires the use of a collection buffer. We also demonstrated that an additional genomic DNA removal step after RNA isolation is required to completely clear the RNA from any contaminating genomic DNA. For cDNA synthesis and library preparation, we combined SmartSeq v4 full length cDNA library amplification, Nextera XT tagmentation and sample barcoding. Using this workflow, we were able to generate highly reproducible RNA sequencing results.ConclusionsThe presented optimized workflow enables to generate high quality RNA and allows accurate transcriptome profiling of small populations of sorted zebrafish cells.

Abstract A137: The innate/adaptive immune response triggered in response to local immunotherapy of orthotopically growing bladder cancer tumors

Abstract The mouse bladder 49 (MB49) syngeneic tumors respond to various immunotherapies, both stimulatory immunotherapies and check-point blockers; when tumors are cured it is often an antitumor memory response established (previous publications by our group and others). Herein we present data suggesting that bladders of orthotopic MB49 tumor-bearing mice hold a reservoir of lymphocytes surrounding the tumor, wherein they migrate rapidly upon immune adjuvant stimulation. The tumors appear to form an accessible immune microenvironment rich in vessels that mediate the homing of leukocytes to the tumor site. This is in contrast to MB49 tumors that grow subcutaneously and do not appear to provide the same rapid immune cell infiltration capacity. Herein we further examined the immune cell populations of tumors locally treated with CpG ODN 1668, a type B CpG and a strong immunostimulatory agent or dilution buffer, as a control. Interestingly, we identified markers of high-endothelial venules (HEVs) in the vessels present in tumors from both untreated and treated animals. Thus we conclude that HEVs develop naturally in the orthotopic MB49 model and we wished to explore whether manipulation of the tumor microenvironment (TME) can induce HEVs and how this stimuli affect the infiltration of immune cells. Immunohistochemical profiling revealed that CD11c+ dendritic cells, CCL21+ cells along with B and T lymphocytes are present in various ways in different zones of the tumor with HEVs and CD31+ vessels. A correlation with vessel density and the presence of HEVs was identified in untreated animals. In immune-stimulated tumors, this correlation was lost in the early phase post stimuli. We could also identify that CpG treated bladder displayed increased vascularization around the tumor 24 hours post treatment. Interestingly, it appeared as CCL21 expression intratumorally aligned with increased recruitment of CD11c+ DCs in the TME. Intratumorally it was a clear correlation between CCL21 expression with both CD11c+ and CD8+ cell infiltration. As CD11c+ dendritic cells migrate towards gradients of CCL21 via their CCR7 receptor, enabling antigen presentation, it is plausible that antigen-presentation takes place in the TME in relation to this finding. By these preliminary data we conclude that in the orthotopic MB49 tumor model, CCL21 appears to play a role in the induction of adaptive immune responses through the CCL21/CCR7 axis. The role of HEVs, or alternatively lymphatic vessels, for this recruitment is yet to be determined. Currently we are investigating multiplex analyses on bladder tumors, urine and blood from tumor-bearing animals to identify cytokines in the TME or systemically that are correlated the formation of an adaptive immune response. Our future goal is to examine what is the key to raise an effective immune response to the so called immune "hot" orthotopic MB49 tumor. Citation Format: Iliana Kyriaki, Sara M. Mangsbo. The innate/adaptive immune response triggered in response to local immunotherapy of orthotopically growing bladder cancer tumors [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A137.

Chromosome Dilution Buffer

Chromosome Dilution Buffer

A Rapid, Sensitive, and Portable Biosensor Assay for the Detection of Botulinum Neurotoxin Serotype A in Complex Food Matrices.

Botulinum neurotoxin (BoNT) intoxication can lead to the disease botulism, characterized by flaccid muscle paralysis that can cause respiratory failure and death. Due to the significant morbidity and mortality costs associated with BoNTs high toxicity, developing highly sensitive, rapid, and field-deployable assays are critically important to protect the nation’s food supply against either accidental or intentional contamination. We report here that the B-cell based biosensor assay CANARY® (Cellular Analysis and Notification of Antigen Risks and Yields) Zephyr detects BoNT/A holotoxin at limits of detection (LOD) of 10.0 ± 2.5 ng/mL in assay buffer. Milk matrices (whole milk, 2% milk and non-fat milk) with BoNT/A holotoxin were detected at similar levels (7.4–7.9 ng/mL). BoNT/A complex was positive in carrot, orange, and apple juices at LODs of 32.5–75.0 ng/mL. The detection of BoNT/A complex in solid complex foods (ground beef, smoked salmon, green bean baby puree) ranged from 14.8 ng/mL to 62.5 ng/mL. Detection of BoNT/A complex in the viscous liquid egg matrix required dilution in assay buffer and gave a LOD of 171.9 ± 64.7 ng/mL. These results show that the CANARY® Zephyr assay can be a highly useful qualitative tool in environmental and food safety surveillance programs.

The Case for Human Serum as a Highly Preferable Sample Matrix for Detection of Anthrax Toxins

This paper describes preliminary results on the surprising impact of human serum as a sample matrix on the detectability of protective antigen (PA) and lethal factor (LF), two antigenic protein markers of Bacillus anthracis, in a heterogeneous immunometric assay. Two sample matrices were examined: human serum and physiological buffer. Human serum is used as a specimen in the diagnostic testing of potentially infected individuals. Physiological buffers are often applied to the recovery of biomarkers dispersed in suspicious white powders and other suspect specimens and as a serum diluent to combat contributions to the measured test response from nonspecific adsorption. The results of these experiments using a sandwich immunoassay read out by surface-enhanced Raman scattering yielded estimates for the limit of detection (LOD) for both markers when using spiked human serum that were remarkably lower than those of spiked physiological buffer (∼70,000× for PA and ∼25,000× for LF). The difference in LODs is attributed to a degradation in the effectiveness of the capture and/or labeling steps in the immunoassay due to the known propensity for both proteins to denature in buffer. These findings indicate that the use of physiological buffer for serum dilution or recovery from a powdered matrix is counter to the low-level detection of these two antigenic proteins. The potential implications of these results with respect to the ability to detect markers of other pathogenic agents are briefly discussed.

Effects of protein size, thermodynamic stability, and net charge on cotranslational folding on the ribosome

During the last five decades, studies of protein folding in dilute buffer solutions have produced a rich picture of this complex process. In the cell, however, proteins can start to fold while still attached to the ribosome (cotranslational folding) and it is not yet clear how the ribosome affects the folding of protein domains of different sizes, thermodynamic stabilities, and net charges. Here, by using arrest peptides as force sensors and on-ribosome pulse proteolysis, we provide a comprehensive picture of how the distance from the peptidyl transferase center in the ribosome at which proteins fold correlates with protein size. Moreover, an analysis of a large collection of mutants of the Escherichia coli ribosomal protein S6 shows that the force exerted on the nascent chain by protein folding varies linearly with the thermodynamic stability of the folded state, and that the ribosome environment disfavors folding of domains of high net-negative charge.

Sperm Dilution Buffer

Sperm Dilution Buffer

Antibody Dilution Buffer (Abdil)

Antibody Dilution Buffer (Abdil)

Strategy to improve the characterization of chitosan by size exclusion chromatography coupled with multi angle laser light scattering

Here we present SEC-MALLS study for chitosan samples with weight average molar mass Mw between 33 and 427 kg/mol on columns packed with 8 μm porous particles. A low injection concentration on the order of 0.1–0.2 mg/mL must be used to avoid overloading of the SEC columns, due to the extended coil conformation of cationic chitosan in the dilute acid buffer as compared to neutral polymers. Additionally, SEC must be performed at an eluent flow rate no more than 0.5 mL/min for high molar mass chitosan samples. At flow rates of 1.0 and 1.5 mL/min, the elution of the largest chitosan species is delayed, which leads to a distortion of the molar mass distribution towards lower molar mass region. Such an abnormal behavior is due to a chromatographic mode transition from SEC to slalom chromatography, originated from the coil–stretch transition of chitosan chains in elongational flow through the packed columns.

Systematic evaluation of the early access applied biosystems precision ID Globalfiler mixture ID and Globalfiler NGS STR panels for the ion S5 system

Systematic evaluation of the early access applied biosystems precision ID Globalfiler mixture ID and Globalfiler NGS STR panels for the ion S5 system