Exclusion Mode Research Articles

Variation of Cyclodextrin (CD) Complexation with Biogenic Amine Tyramine: Pseudopolymorphs of β-CD Inclusion vs. α-CD Exclusion, Deep Atomistic Insights.

Tyramine (TRM) is a biogenic catecholamine neurotransmitter, which can trigger migraines and hypertension. TRM accumulated in foods is reduced and detected using additive cyclodextrins (CDs) while their association characteristics remain unclear. Here, single-crystal X-ray diffraction and density functional theory (DFT) calculation have been performed, demonstrating the elusive pseudopolymorphs in β-CD inclusion complexes with TRM base/HCl, β-CD·0.5TRM·7.6H2O (1) and β-CD·TRM HCl·4H2O (2) and the rare α-CD·0.5(TRM HCl)·10H2O (3) exclusion complex. Both 1 and 2 share the common inclusion mode with similar TRM structures in the round and elliptical β-CD cavities, belong to the monoclinic space group P21, and have similar herringbone packing structures. Furthermore, 3 differs from 2, as the smaller twofold symmetry-related, round α-CD prefers an exclusion complex with the twofold disordered TRM-H+ sites. In the orthorhombic P21212 lattice, α-CDs are packed in a channel-type structure, where the column-like cavity is occupied by disordered water sites. DFT results indicate that β-CD remains elliptical to suitably accommodate TRM, yielding an energetically favorable inclusion complex, which is significantly contributed by the β-CD deformation, and the inclusion complex of α-CD with the TRM aminoethyl side chain is also energetically favorable compared to the exclusion mode. This study suggests the CD implications for food safety and drug/bioactive formulation and delivery.

New mode of HPLC separation for multi‐charged polymers, peptides, and proteins

AbstractThis article explores a new mode of chromatographic separation named BIST™ (Bridge Ion Separation Technique) that is designed specifically for the retention and separation of charged molecules. The proposed mechanism is supported by multiple examples. The separation technique is especially useful for analysis of low molecular weight charged polymers, the class of molecules where ion‐exchange, ion‐exclusion and size exclusion modes of separation do not work effectively. Among low molecular weight polymers, many compounds are of high importance for biological, medicinal, agricultural, and consumer applications. Some examples include heparin, poly amino acids, insulin, polyethyleneimine, as well as other peptides. The wide variety of applications of many charged polymers necessitates a high‐quality analytical technique for testing and research of these compounds.

Top-Down Mass Spectrometry of Synthetic Single Guide Ribonucleic Acids Enabled by Facile Sample Clean-Up.

In recent years, CRISPR-Cas9 genome editing has become an important technology in biomedical research and has demonstrated tremendous therapeutic potential. With Cas9 endonuclease, the use of single guide ribonucleic acids (sgRNAs) allows for sequence-specific cutting on target double-stranded deoxyribonucleic acids. Therefore, the design and quality of sgRNAs can greatly affect the efficiency and specificity of genome editing. Mass spectrometry (MS) has been a powerful tool to detect molecular features and sequence a variety of biomolecules; however, as the sizes of oligonucleotides get larger, it becomes more challenging to desalt samples and achieve high-quality intact spectra with effective fragmentation. Here, we develop a simple but effective online column-based clean-up method (reversed-phase column in a size exclusion mode) that removes formulation salts and metal adducts from larger oligonucleotides upon entering the mass spectrometer in a consistent manner. Using the top-down approach without any nuclease digestion, we characterized and sequenced 100-nucleotide-long sgRNAs by higher-energy collision dissociation (HCD), collision-induced dissociation (CID), ultraviolet photodissociation (UVPD), and activated electron photodetachment (a-EPD). In a single 10 min liquid chromatography-tandem MS (LC-MS/MS) run, CID yielded the best sequence coverage, of 67%. When adding complementary UVPD and a-EPD runs, we achieved 80% overall sequence coverage and 100% cleavages for the variable sequence, the first 20 nucleotides from the 5' end. This LC-MS/MS platform provides a facile top-down workflow to analyze and sequence larger chemically modified oligonucleotides with no sample treatment.

Simulador para la evaluación de algoritmos para la gestión de recursos compartidos en sistemas distribuidos

In distributed processing systems, especially when processes constitute collaborative groups, it is necessary that, when allocating resources to processes, decisions are made based on agreements regarding access to resources; decisions may be related to the performance of a certain activity that requires or not the synchronization of processes, requiring the use of shared resources in the mode of mutual exclusion. Thus, the following question arises: What are the decision models and aggregation operators that will have to be generated by incorporating the global cognitive perspective to the classical models to make more intelligent decision making in the management of resources and groups of processes taking into account self-regulation? How will the algorithms of the different decision models be implemented? How to validate the new proposed algorithms by comparing them with each other and with traditional algorithms? For this, it is necessary to have a simulator that implements the traditional and the new proposed algorithms and allows to observe their behavior and results under different types of workloads. Classical models are considered for accessing shared resources in the mutual exclusion mode using critical regions to the centralized algorithm, the distributed algorithm of Lamport, Ricart and Agrawala, the token ring algorithm, among others. A prototype simulator is presented to evaluate the performance of the proposed new decision models and aggregation operators against the main traditional models.

Size exclusion chromatography of petroleum samples using amino-bonded silica phase

Amino-bonded silica phase, frequently used in adsorption chromatographic mode for petroleum analysis, was evaluated for the separation of petroleum components based on polymer liquid chromatographic modes (viz. size exclusion, critical condition, and interaction). After verification of the stationary phase for polymer chromatography using polystyrene standards at different compositions of cyclohexane and dichloromethane, a similar approach was attempted for a distilled fraction of a crude oil. The approach was successful in size exclusion chromatographic mode whereas other modes were partially achievable because of the complex nature of petroleum samples. Subsequently, size exclusion mode was applied in crude oils with different American Petroleum Institute (API) gravity values. An inverse linear relationship between the API gravity value and the average molecular weights of the aromatic compounds of crude oils was established. It was found that the weight average molecular weight (Mw) was more sensitive to changes in API gravity values of the crude oils in comparison to number average molecular weight (Mn). Additionally, crude oil with lower API gravity value reflected broader distribution of species. Further studies on crude oils with lower API gravity value (<20°) and of different geographical origins will confirm the universality of the method. This method will provide reliable average molecular weights of the aromatic content for aromatic rich refinery streams to optimize process conditions. Furthermore, the developed method can potentially be extended to a two-dimensional LC method using the same stationary phase with different mobile phase compositions for comprehensive characterization of petroleum samples.

Photoresistors from heteroepitaxial structures n-CdxHg1-xTe with exclusion of charge carriers within the 3–5 µm spectral range

The properties of photoresistors with photosensitive areas of 0.05mm×0.05mm and 0.5mm×0.5mm made of heteroepitaxial structures n-CdxHg1−xTe were investigated within the spectral range of 3–5 µm using indium contacts at a temperature of 80 K. The dependence of the lifetime and concentration of the charge carriers inside the pixels on the bias voltage was determined within a wide range of exposure to background radiation. In photoresistors with a pixel size of 0.05mm×0.05mm, a nonequilibrium mode of operation with the exclusion of minority charge carriers can be realized. The electron concentration in this mode decreases to 1×1013cm−3. Photoresistors made of epitaxial structures n-CdxHg1−xTe (x≈0.305) in exclusion mode at 80 K have a voltage sensitivity of the order of 107V/W and a specific detectivity of approximately 1.3×1012cm⋅Hz1/2/W.

Importance of Particle Pore Size in Determining Retention and Selectivity in Reversed Phase Liquid Chromatography

Column selection often centers on the identification of a stationary phase that increases resolution for a certain class of compounds. While gains in resolution are most affected by selectivity of the stationary phase or modifications of the mobile phase, enhancements can still be made with an intentional selection of the packing material's microstructure. Unrestricted mass transfer into the particle's porous structure minimizes band broadening associated with hindered access to stationary phase. Increased efficiency, especially when operating above the optimal flow rates, can be gained if the pore size is significantly larger than the solvated analyte. Less studied are the effects of reduced access to pores due to physical hindrance and its impact on retention. This article explores the relationship between pore size and reversed phase retention, and specifically looks at a series of particle architectures with reversed phase and size exclusion modes to study retention associated with access to stationary phase surface area.

Fault-Resilient Continuum Deformation Coordination

This article offers a physics-based automation with two operation modes: 1) homogeneous deformation mode (HDM) and 2) containment exclusion mode (CEM), to safely plan a large-scale group coordination and resiliently recover safety in the presence of unpredicted agent failures. HDM becomes active when all agents are healthy, where the group coordination is defined by homogeneous transformation coordination functions. At HDM, a desired n-D homogeneous transformation (n = 2, 3) is uniquely related to the desired trajectories of n + 1 leaders and acquired by the remaining followers in real-time through local communication. At CEM, agent coordination is treated as an ideal fluid flow, where the desired agents' paths are defined along stream lines inspired by fluid flow field theory to circumvent exclusion spaces surrounding failed agent(s). This article formally specifies the transitions between the HDM and CEM by using local proximity and applying the continuum deformation properties. More specifically, local proximity is used by agents to define interagent communication in an unsupervised fashion once they recover safety and HDM is activated. On the other hand, properties of homogeneous transformation are applied to quickly detect each arising anomalous situation and exclude failed agent(s) from group coordination of healthy agents.

Ion exchange and ion exclusion chromatographic characterization of wines using conductivity detection

High performance ion chromatography system employing ion exchange and ion exclusion modes was used for the separation and quantitative analysis of tartarate, malate, citrate, lactate, chloride, nitrate, phosphate, sulfate, lithium, sodium, potassium, magnesium, and calcium in 15 wines originated worldwide. All anions were measured by means of conductivity detection. Analytical figures of merit were assessed and reported for all analyzed ions. In addition, the classic physicochemical properties of wines, i.e. color, acidity and density, were determined. Correlations between measured variables were evaluated and discussed. The proposed procedure is exceptionally simple and requires only regular instrumentation; nevertheless, it provides possibility for fast, straightforward and sensitive quantification of aforementioned ions. Sample preparation required only filtration and dilution where necessary.

Characterization of polyethermethylsiloxanes using ultra-high performance liquid chromatography-electrospray ionization and time-of-flight mass spectrometry

Polyethermethylsiloxanes (PEMSs) are silicone based polymers formed by attaching one or more ethylene oxide (EO)/propylene oxide (PO) chains to a siloxane chain. As the siloxane chain is lengthened, the polarity of the PEMSs are reduced. Little research has been conducted on the use of ultra-high performance liquid chromatography (UHPLC)-mass spectrometry (MS) to analyze PEMS oligomers with more than 4 siloxane groups because of their low polarity and poor solubility in water and acetonitrile (ACN). In this study, we developed a high chromatographic resolution method for PEMS and polyether oligomers using the water-ACN-isopropanol (IPA) tertiary solvents gradient. PEMSs oligomers containing one EO pendant chain and 3–13 silicone groups were analyzed. More than 102 PEMS oligomers and 21 polyether oligomers were separated within 45 min and identified by accurate molecular masses. During this gradient elution process, different chromatographic modes were used: both precipitation-redissolution and adsorption mode for PEMSs, adsorption mode for EO chains in polyethers, and exclusion mode for EO chains in PEMSs. This efficient separation method for PEMSs would broaden the characterization of silicone surfactants. Also, it is beneficial to establish the relationship between surfactant structure, synthetic route and performance optimization.

Swine-Derived Probiotic Lactobacillus plantarum Inhibits Growth and Adhesion of Enterotoxigenic Escherichia coli and Mediates Host Defense.

Weaning stress renders piglets susceptible to pathogen infection, which leads to post-weaning diarrhea, a severe condition characterized by heavy diarrhea and mortality in piglets. Enterotoxigenic Escherichia coli (ETEC) is one of typical strains associated with post-weaning diarrhea. Thus, prevention and inhibition of ETEC infection are of great concern. Probiotics possess anti-pathogenic activity and can counteract ETEC infection; however, their underlying mechanisms and modes of action have not yet been clarified. In the present study, the direct and indirect protective effects of Lactobacillus plantarum ZLP001 against ETEC infection were investigated by different methods. We found that bacterial culture and culture supernatant of L. plantarum ZLP001 prevented ETEC growth by the Oxford cup method, and ETEC growth inhibition was observed in a co-culture assay as well. This effect was suggested to be caused mainly by antimicrobial metabolites produced by L. plantarum ZLP001. In addition, adhesion capacity of L. plantarum ZLP001 to IPEC-J2 cells were observed using microscopy and counting. L. plantarum ZLP001 also exhibited a concentration-dependent ability to inhibit ETEC adhesion to IPEC-J2 cells, which mainly occurred via exclusion and competition mode. Furthermore, quantitative real time polymerase chain reaction (qPCR) analysis showed that L. plantarum ZLP001 upregulated the expression of host defense peptides (HDPs) but did not trigger an inflammatory response. In addition, L. plantarum ZLP001 induced HDP secretion, which enhanced the potential antimicrobial activity of IPEC-J2 cell-culture supernatant after incubation with L. plantarum ZLP001. Our findings demonstrate that L. plantarum ZLP001, an intestinal Lactobacillus species associated with piglet health, possesses anti-ETEC activity. L. plantarum ZLP001 might prevent ETEC growth, inhibit ETEC adhesion to the intestinal mucosa, and activate the innate immune response to secret antimicrobial peptides. L. plantarum ZLP001 is worth investigation as a potential probiotics.

Role of the Excluded Strand in DNA Unwinding by Hexameric Helicases

Hexameric DNA helicases control both the initiation and elongation phases of DNA replication. It is well known that these toroidal enzymes encircle and translocate preferentially on one strand with specific polarities dependent on their families and organismal domains. The question remains as to what if any role the excluded strand has in the unwinding mechanism or coordination of activities at the replication fork. Over the past few years, there has been an increasing amount of evidence to suggest that the excluded‐strand plays a significant role during unwinding by interacting with the helicase's outer surface in a Steric Exclusion and Wrapping (SEW) mode. The implications are that the excluded strand interaction controls the kinetics of unwinding while maintaining the ssDNA product for handoff to other replication components.We have investigated the interaction of the excluded strand with the outer surface of helicases in archaea, mitochondria, and bacteria using a variety of biochemical, biophysical, and genetic techniques. We have not only defined a minimal interaction path on the outer surface of these helicases, but we can also show that this interaction differentially controls the unwinding rate and efficiency. For the archaea MCM helicase, which has 3′‐5′ polarity, disruption of the excluded strand interaction inhibits unwinding consistent with a ratchet mechanism. Conversely, for the bacterial DnaB helicase, which has 5′‐3′ polarity, disruption increases the unwinding rate more consistent with a regulator‐type mechanism. The contrasting effects of the excluded strand interaction coupled with the opposing translocation polarities of these helicases will be discussed within the context of their respective replisomes.Support or Funding InformationNSF‐1613534

Inhibition of Streptococcus mutans Growth and Biofilm Formation by Probiotics in vitro

To exert anticaries effects, probiotics are described to inhibit growth and biofilm formation of cariogenic bacteria such as Streptococcus mutans (SM). We screened 8 probiotics and assessed how SM growth or biofilm formation inhibition affects cariogenicity of probiotic-SM mixed-species biofilms in vitro. Growth inhibition was assessed by cocultivating probiotics and 2 SM strains (ATCC 20532/25175) on agar. Probiotics were either precultured before SM cultivation (exclusion), or SM precultured prior to probiotic cultivation (displacement). Inhibition of SM culture growth was assessed visually. Inhibition of SM biofilm formation on bovine enamel was assessed using a continuous-flow short-term biofilm model, again in exclusion or displacement mode. The cariogenicity of mixed-species biofilms of SM with the most promising growth and biofilm formation inhibiting probiotic strains was assessed using an artificial mouth model, and enamel mineral loss (ΔZ) was measured microradiographically. We found limited differences in SM growth inhibition in exclusion versus displacement mode, and in inhibition of SM 20532 versus 25175. Results were therefore pooled. Lactobacillus acidophilus LA-5 inhibited significantly more SM culture growth than most other probiotics. L. casei LC-11 inhibited SM biofilm formation similarly to other alternatives but showed the highest retention of probiotics in the biofilms (p < 0.05). Mineral loss from SM monospecies biofilms (ΔZ = 9,772, 25th/75th percentiles: 6,277/13,558 vol% × µm) was significantly lower than from mixed-species SM × LA-5 biofilms (ΔZ = 24,578, 25th/75th percentiles: 19,081/28,768 vol% × µm; p < 0.01) but significantly higher than from SM × LC-11 biofilms (ΔZ = 4,835, 25th/75th percentiles: 263/7,865 vol% × µm; p < 0.05). Probiotics inhibiting SM culture growth do not necessarily reduce the cariogenicity of SM-probiotic biofilms. Nevertheless, SM biofilm formation inhibition may be relevant in the reduction of cariogenicity.

Problems in the size exclusion chromatography of poly(N-isopropylacrylamide) on styragel columns

The molecular weights of poly(N-isopropylacrylamide) (PNIPA), calculated according to polystyrene calibration standards upon the elution of THF on styragel columns, appear to be much lower than their actual values determined using independent approaches. This is likely due to interactions between the nitrogen-containing units of PNIPA polymer chains and the sorbent, so the polymer is eluted in the mode intermediate between exclusion and critical. An effective exclusion mode during the elution of PNIPA on a styragel column can be achieved by using an eluent more polar than tetrahydrofuran (particularly, 1-methylpyrrolidone).

Estimation of critical conditions of polymers based on monitoring the polymer recovery

Liquid chromatography at critical conditions (LCCC) is a very attractive chromatographic technique on the border between the size exclusion and liquid adsorption mode of the liquid chromatography. The strong interest in LCCC arises from the fact that it is well suited to analyze the block lengths in segmented copolymers or the heterogeneities with regard to end groups present, for example, in functionalized polymers e.g., telechelics. In this paper a new method for identification of the critical conditions of synthetic polymers is proposed, which requires only one polymer sample with higher molar mass. The method is based on monitoring the recovery of the polymer sample from a column. The composition of the mobile phase is modified until the polymer sample is fully recovered from the column. The corresponding composition of the mobile phase is composition corresponding to LCCC. This new method was applied for the determination of critical conditions for polyethylene, syndiotactic polypropylene and isotactic polypropylene. The results of the new method will be compared to those of classical approaches and advantages will be pointed out.

Water ICE: Ion Exclusion Chromatography of Very Weak Acids with a Pure Water Eluent.

Separation of ions or ionizable compounds with pure water as eluent and detecting them in a simple fashion has been an elusive goal. It has been known for some time that carbonic acid can be separated from strong acids by ion chromatography in the exclusion mode (ICE) using only water as the eluent. The practice of water ICE was shown feasible for very weak acids like silicate and borate with a dedicated element specific detector like an inductively coupled plasma mass spectrometer (ICPMS), but this is rarely practical in most laboratories. Direct conductometric detection is possible for H2CO3 but because of its weak nature, not especially sensitive; complex multistep ion exchange methods do not markedly improve this LOD. It will clearly be impractical in acids that are weaker still. By using a permeative amine introduction device (PAID, Anal. Chem. 2016 , 88 , 2198 - 2204 ) as a conductometric developing agent, we demonstrate that a variety of weak acids (silicate, borate, arsenite, cyanide, carbonate, and sulfide) cannot only be separated on an ion exclusion column, they can be sensitively detected (LODs 0.2-0.4 μM). We observe that the elution order is essentially the same as that on a nonfunctionalized poly(styrene-divinylbenzene) column using 1-10% acetonitrile as eluent and follows the reverse order of the polar surface area (PSA) of the analyte molecules. PSA values have been widely used to predict biological transport of pharmaceuticals across a membrane but never to predict chromatographic behavior. We demonstrate the application of the technique by measuring the silicate and borate depth profiles in the Pacific Ocean; the silicate results show an excellent match with results from a reference laboratory.

Bacteriocin-producing strains of Lactobacillus plantarum inhibit adhesion of Staphylococcus aureus to extracellular matrix: quantitative insight and implications in antibacterial therapy

In the present study, the adhesion of bacteriocin-producing probiotic strains of Lactobacillus plantarum onto extracellular matrix (ECM) proteins such as collagen and mucin and their potential to prevent pathogen invasion onto the ECM was ascertained. Fluorescence-based in vitro assays indicated that L. plantarum strains CRA21, CRA38 and CRA52 displayed considerable adhesion to ECM molecules, which was comparable to the probiotic Lactobacillus rhamnosus GG. Flow cytometry-based quantitative assessment of the adhesion potential suggested that L. plantarum CRA21 exhibited superior adhesion onto the ECM as compared with other lactic acid bacteria strains. Furthermore, fluorescence-based assays suggested that the highest inhibition of Staphylococcus aureus adhesion onto collagen and mucin by bacteriocin-producing L. plantarum strains was observed in the exclusion mode as compared with the competition and displacement modes. This observation was supported by the higher binding affinity (k(d)) for the ECM exhibited by the L. plantarum strains as compared with S. aureus. Interestingly, a crude plantaricin A extract from food isolates of L. plantarum displayed potent antibacterial activity on ECM-adhered S. aureus cells. It is envisaged that the L. plantarum isolates displaying bacteriocinogenic and ECM-adhering traits can perhaps be explored to develop safe antibacterial therapeutic agents.

Utilization of a precolumn with size exclusion and reversed-phase modes for size-exclusion chromatographic analysis of polysorbate-containing protein aggregates

Size-exclusion chromatography (SEC) is a useful method for quantification of protein aggregates because of its high throughput capacity and highly quantitative performance. One of the problems in this method concerns polysorbates, which are well-known additives for protein-containing products to prevent protein aggregation, but frequently interfere with the photometric detection of protein aggregates. We developed a new SEC method that can separate polysorbates from protein sample solutions in an on-line mode with a precolumn with size exclusion and reversed-phase mixed modes. The precolumn can effectively trap polysorbates in aqueous mobile phase, and the trapped polysorbates are easily eluted with acetonitrile-containing aqueous mobile phase to clean the precolumn. Small parts of protein aggregates may be also trapped on the precolumn depending on temperature and proteins. Setting appropriate column temperature can minimize such inconvenient trapping of aggregates.

HPLC Characterization of Hydrogenous Polystyrene-block-deuterated polystyrene Utilizing the Isotope Effect

HPLC elution behavior of isotopically different block copolymers was investigated. A series of three diblock copolymers of hydrogenous polystyrene and deuterated polystyrene (hPS-b-dPS), in which the length of hPS-block is fixed at 18 kg/mol and the length of dPS-block is varied from 17 kg/mol to 80 kg/mol, was synthesized and their retention behavior in liquid chromatography at critical condition (LCCC) was investigated using a C18 coated silica stationary phase and a mixed solvent of CH2Cl2 and CH3CN. At this LC separation condition, hPS is retained slightly longer than dPS. The LCCC separations were performed at both LCCC conditions of dPS and hPS established with the same stationary and mobile phases, but at different column temperatures. Since the chromatographic retention difference between dPS and hPS is very small, it was possible to elute the block copolymers at both exclusion and interaction modes at the critical condition of each individual block. We found that the block at its critical condition is not fully invisible in both cases. In the LCCC separation at the critical condition of dPS, hPS-b-dPS elutes after the injection solvent peak (interaction mode) due to the stronger interaction of the hPS block. Although they have the same hPS block, they do not coelute but elute in the order of decreasing total molecular weight like an elution in exclusion mode. It clearly demonstrates that the dPS block is not invisible at the critical condition but influences the retention of the block copolymers. Monte Carlo simulations of the partition coefficient of A-b-B into a slit pore were performed to give insight on the elution behavior of A-b-B at the critical condition of the B block. The simulation shows that a block under the critical condition influences the retention of the other visible block whether the visible block is eluted in exclusion or interaction mode. When A is eluted in the interaction mode and B is at its critical condition, the partition coefficient is found to decrease with the increase of the invisible B block length, conforming to the observed elution behavior in experiments.

Determination of Caprolactam and Residual Vinyl Caprolactam Monomer in Soluplus by Mixed Mode Gel Permeation Chromatography

Soluplus, a graft copolymer of polyethylene glycol, vinyl caprolactam and vinyl acetate, is designed to solubilize poorly soluble active pharmaceutical ingredients. A straightforward aqueous gel permeation chromatography method that exploits both size exclusion and adsorption modes of separation was used to separate and quantify the related residual vinyl caprolactam monomer and caprolactam impurity present in Soluplus. This methodology offers a single step analysis of caprolactam and the residual vinyl caprolacatam monomer, yielding similar results to reversed-phase chromatography measurements, which are time-consuming and may involve multi-step sample preparation. The results of this study demonstrate that gel permeation chromatography provides a viable option to traditional reversed-phase chromatography in the quantitative analysis of residual caprolactam and vinyl caprolactam monomers and can be extended to other monomer-polymeric systems.