Column Calibration Research Articles

Investigating molecular markers linked to acute myocardial infarction and cuproptosis: bioinformatics analysis and validation in the AMI mice model.

Cuproptosis-related key genes play a significant role in the pathological processes of acute myocardial infarction (AMI). However, a complete understanding of the molecular mechanisms behind this participation remains elusive. This study was designed to identify genes and immune cells critical to AMI pathogenesis. Based on the GSE48060 dataset (31 AMI patients and 21 healthy persons, GPL570-55999), we identified genes associated with dysregulated cuproptosis and the activation of immune responses between normal subjects and patients with a first myocardial attack. Two molecular clusters associated with cuproptosis were defined in patients with AMI. Immune infiltration analysis showed that there was significant immunity heterogeneity among different clusters. Multiple immune responses were closely associated with Cluster2-specific differentially expressed genes (DEGs). The generalized linear model machine model presented the best discriminative performance with relatively lower residual and root mean square error, and a higher area under the curve (AUC = 0.870). A final two-gene-based generalized linear model was constructed, exhibiting satisfactory performance in two external validation datasets (AUC = 0.719, GSE66360 and AUC = 0.856, GSE123342). Column graph, calibration curve, and decision curve analyses also proved the accuracy of AMI prediction. We also constructed a mouse C57BL/6 model of AMI (3 h, 48 h, and 1 week) and used qRT-PCR and immunofluorescence to detect the expression changes of CBLB and ZNF302. In this study, we present a systematic analysis of the complex relationship between cuproptosis and a first AMI attack, and provide new insights into the diagnosis and treatment of AMI.

Resonant Column Calibration and Dynamic Torsional Shear Testing Using Stepped Frequency Sweeps

Abstract This article introduces a new apparatus calibration procedure along with testing protocols, data acquisition, and reduction procedures for a fixed-free resonant column test in which a fixed amplitude sinusoidal torque is applied to the specimen at discrete frequencies over a wide bandwidth that encompasses the resonant frequency. Recently developed resonant column theory, consistent with ASTM D4015-21, Standard Test Methods for Modulus and Damping of Soils by Fixed-Base Resonant Column Devices, is shown to hold for all frequencies in this bandwidth and enables determination of shear modulus, damping, and shear strain amplitude at each frequency. For off-resonant frequencies, shear strain amplitudes are much lower than those at resonance; hence, under a constant applied torque, the method measures values over a range of shear strains. These procedures are facilitated using a spectrum analyzer. The new and more robust approach for apparatus calibration relies on conducting stepped frequency sweeps (SFS) on calibration rods and allows concurrent determination of all apparatus calibration factors, which apply to the full frequency bandwidth for testing soils. Test results on sand are used to illustrate the developed procedures and recommendations are provided for determining shear modulus and damping over the full shear strain range of the apparatus. If the fixed amplitude applied torque is low, the test provides shear modulus and damping at very low strains. For sufficiently high values of the applied torque, a single SFS allows semicontinuous characterization of the shear modulus degradation and shear damping increase behavior. The article provides recommendations for selecting frequency bandwidth, frequency step values, and spectrum analyzer settings. For testing with large input torques, SFS should be from high frequency to low frequency to more accurately capture the response function for strain softening behavior. The total time for SFS is on the order of 10 min, and the number of applied loading cycles can be controlled.

Densification of activated sludge for better settling performances: experimental characterization in batch column and model parameters calibration.

Conventional activated sludge (CAS) and densified sludge obtained using hydro-cyclone selective wasting were compared at a full-scale water resources recovery facility. The densified tested sludge, containing around 30-50% of aerobic granules, showed enhanced settleability with low and stable sludge volume index (SVI) compared to CAS, which suffered recurrent filamentous bulking. Further in-depth batch settling tests were carried out using a 40 cm diameter column fitted with ultrasonic transducers to monitor both sludge blanket height and vertical velocity profiles. Hindered settling and compression parameters were calibrated from the experiment for latter modelling use. Test sludge displayed more than doubled settling velocities compared to CAS, with hindered settling velocities remaining >3 m·h-1 even at high solids concentrations of 6.85 g·L-1. The compression regime was attained at much higher critical concentration for the test sludge. It also displayed enhanced thickening properties, with concentrations obtained after 30 min of settling being 20.9 and 8.5 g·L-1 respectively for test and control sludge. This allows for a substantial reduction of recirculation rates in practice. These results open perspectives in optimizing existing plant operation as well as clarifier design and modelling using densified sludge.

A reliability-based approach and code calibration of FRP-Confined rectangular RC columns subjected to concentric loading

This study aims to present an approach to investigate the reliability levels of reinforced concrete (RC) columns confined by Fiber Reinforced Polymers (FRP) subjected to concentric loading. First, the statistical properties of random variables such as model error, FRP strain efficiency factor (FSE factor) and axial resistance of columns are investigated. Then in respect to the existing correlation between variables and extensive collected database, an appropriate probabilistic model for model error, FSE factor and compressive resistance of RC column is proposed. In addition, the effects of live-to-dead load ratio, reinforcement ratio and the confinement levels on the reliability of FRP-confined columns are studied through a reliability analysis. Furthermore, based on existing studies, reliability indices for various levels of confinement are calculated. Finally, a calibration analysis on safety factors of ACI 440.2R-17 guideline and shape factor of rectangular cross-section is presented.

Size distribution and chain conformation of six different fucoidans using size-exclusion chromatography with multiple detection

Fucoidans represent an intriguing class of fucose-containing sulfated polysaccharides. The biological activities of these polysaccharides are related to their compositional and structural parameters, whereby their degree of sulfation, as well as molecular weight (MW) distribution and chain conformation play important roles. Modern NMR and mass spectrometry techniques allow elucidating details of the glycan structure, but not the structure of the whole molecules in their native state. Accordingly, the knowledge about the latter of the fucoidans is currently still limited. Contrary to traditional MW determination by SEC with column calibration, SEC with triple detection provides not only the absolute Mw, but can also give information on additional molecule characteristics. In the present study, we used this method to compare six fucoidans extracted from Fucus vesiculosus (FV), F. serratus (FS), F. evanescens (FE), Dictyosiphon foeniculaceus (DF), Laminaria digitata (LD), and Saccharina latissima (SL) concerning their molar mass (Mw, Mn, Mp, dispersity) and size (rms radius, Rh) characteristics and distribution as well as their chain conformation in solution. The tested fucoidans displayed considerable structural diversity including large differences in their MW profiles and showed to be heterogeneously composed. Fuc-FV and Fuc-SL showed the broadest MW distributions, those from Fuc-FE and Fuc-DF the narrowest ones. Most of the fucoidan fractions (except for Fuc-DF) turned out to exist as expanded flexible chains in PBS solution. The conformation data suggest branched structures with partly long side chains. The knowledge obtained by this study is useful for further fractionation and structural characterization as well as the interpretation of the bioactivity differences between the various fucoidans.

Multi‐Angle Light Scattering: An Efficient Tool Revealing Molecular Structure of Synthetic Polymers

Multi‐angle light scattering (MALS) photometers represent the most effective solution of the traditional problem of size‐exclusion chromatography (SEC), which is column calibration by means of narrow polymer standards. In addition to the ability to provide true molar mass distribution independently of any polymer standards, the combined SEC‐MALS technique is able to yield other important characteristics of polymers such as chain conformation and branching. Coupling an SEC‐MALS set‐up with an online viscosity detector is another step forward to the detailed insight into the polymer molecular structure. Asymmetric flow filed flow fractionation (AF4) is an alternative analytical separation technique capable of replacing SEC in the case of its failure. The paper provides a brief overview of various applications of SEC‐MALS and AF4‐MALS in the R&D and quality control of industrial polymers including introduction into basic theoretical principles.

Sequential Injection Ion Chromatography with Flow Injection Post Column Derivatization Capable of Using the Unstable Reagent Murexide to Determine Calcium and Magnesium in a Mixture

A hyphenated sequential injection ion chromatography–flow injection analysis (SIIC–FIA) system is proposed for post column derivatization capable of using murexide, an unstable but commonly available reagent, for the determination of calcium and magnesium in a mixture. A short ion exchange column was modified from a 4.6 mm × 10 mm C18 monolithic guard column. Calcium/magnesium mixture and murexide were used as model analytes and derivatizing agent, respectively. The FIA post column allows the use of unstable chromogenic reagents. The continuous flow of liquid also helped to stabilize the signal baseline during detection. A single concentration based calibration graph was demonstrated. The system requires low volumes of standard/sample (5–50 µL) and mobile phase (4.50 mL), and offers sample throughput at 8 h−1 without the need for long column calibration. Detection limits were 0.5 and 0.1 µg for Ca2+ and Mg2+, respectively. Working ranges were 1–5 µg Ca2+ and 0.2–1 µg Mg2+. Analyses of well and drinking water samples were demonstrated and validated with the standard complexometric titration.

Size-exclusion chromatography of xylan derivatives-the critical evaluation of macromolecular data.

Hydroxypropyl xylans with varying degrees of substitution were characterized by size-exclusion chromatography. Molar masses of the samples were determined using two approaches: by conventional calibration with molar mass standards and by a multi-detection method that utilizes the combination of static light scattering, viscometry, and differential refractive index detection. The molar mass results obtained by the multi-detection method were accurate, but required the determination of separate refractive index increments for each structurally different sample. The column calibration approach with standard pullulan samples gave biased results due to the differences in hydrodynamic volumes between pullulans and hydroxypropyl xylans with similar molar masses. The degree of hydroxypropylation affected the chain conformation and compactness of the polymer chains. Mark-Houwink parameters and persistence length values suggested that the hydroxypropyl substituents reduced the flexibility of the xylan chain and made the polymer chain more extended.

Abstract 2615: Dietary flavonoid fisetin increases abundance of high-molecular-mass hyaluronan conferring resistance to prostate oncogenesis

Abstract With an estimated 220,800 new cases and 27,450 deaths in 2015, prostate cancer (PCa) continues to be the second leading cause of cancer related deaths in men in the US alone. Accumulation and turnover of extracellular matrix (ECM) is a hallmark of tissue injury, repair and remodeling in human diseases including PCa. Hyaluronan (HA), an extracellular glycosaminoglycan, is a major component of the ECM and plays an important role in regulating reepithelization, tissue healing and wound repair. Elevated HA levels have been shown to be associated with aggressiveness and disease progression in certain cancer types including PCa. Biological responses triggered by HA mainly depend on the HA polymer length. High molecular mass (HMM)-HA represses mitogenic signaling and has anti-inflammatory properties while low molecular mass (LMM)-HA is pro-angiogenic and promotes proliferation and inflammation. Diet-derived agents, such as flavonoids, are of particular interest for cancer chemoprevention as they offer a relatively favorable safety profile. Using HPLC-MS analysis, we identified HA as a novel target of the dietary flavonoid fisetin in tumor xenografts of human PCa NB11 and NB26 cells. Fisetin treatment decreased HA levels in PCa cells both in-vitro (40μM:40h) and in-vivo (1mg/animal). We then evaluated the effect of intraperitoneal administration of fisetin on HA size accumulation and prostate carcinogenesis using the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. Using the combination of Size Exclusion Chromatography (SEC) with Multi-Angle Laser Light Scattering (MALS) analysis that overcomes limitations of column calibration, we evaluated intracellular and extracellular molar mass distribution of HA in fisetin treated cell culture and animal models. We observed lower levels of HMM-HA and higher levels of pro-angiogenic LMW-HA fragments in human PCa PC3 cells. In contrast, we found that fisetin treatment increased HMM-HA and reduced LMM-HA levels in PCa cells. Remarkably, the HA size profile of fisetin treated cells resembled those of normal RWPE1 cells, which predominantly exhibited higher abundance of anti-angiogenic HMM-HA and no detectable LMW-HA. Likewise, secreted HA in fisetin treated PCa cells comprised predominantly of LMM-HA, suggesting greater accumulation of anti-angiogenic HMM-HA within the cells. Molar mass analysis of fisetin treated TRAMP tumor tissues showed an abundance of HMM-HA and reduced levels of LMM-HA when compared to untreated controls. Similarly, secreted HA in fisetin treated TRAMP animals exhibited increased pool of pro-angiogenic LMM-HA, suggesting greater accumulation of anti-angiogenic HMM-HA within the tissues. Our findings suggest that fisetin confers resistance to PCa oncogenesis by increasing the abundance of anti-angiogenic HMM-HA which is associated with delayed disease progression. Citation Format: Rahul K. Lall, Deeba N. Syed, Mohammad Imran Khan, Vaqar M. Adhami, Yuansheng Gong, John A. Lucey, Hasan Mukhtar. Dietary flavonoid fisetin increases abundance of high-molecular-mass hyaluronan conferring resistance to prostate oncogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2615.

Gas chromatography for in situ analysis of a cometary nucleus V. Study of capillary columns’ robustness submitted to long-term reduced environmental pressure conditions

With the European Space Agency's Rosetta space mission to comet 67P/Churyumov-Gerasimenko, a gas chromatograph, part of the COmetary Sampling And Composition (COSAC) experiment, travelled for about 10 years in the interplanetary medium before operating at the surface of the cometary nucleus in November 2014. During its journey in space, the instrument was exposed to the constraining conditions of the interplanetary medium, including reduced environmental pressures. In order to estimate the potential influence of this severe condition on the chromatographic capillary columns, their stationary phase and the subsequent separation capability, a set of flight spare columns were kept under reduced environmental pressure in the laboratory for the same duration as the probe sent to the comet. The columns’ analytical performances were evaluated recently and compared to the original ones obtained just before the launch of the Rosetta probe. The results presented here show that the chromatographic performances of the spare chromatographic columns were not altered in time. From this result, it can be expected that the flight instrument will perform nominally for the analysis of the first cometary nucleus sample to be collected ever, and that the preparation of the interpretation of the data to be taken at the cometary surface nucleus can be done through calibration of these spare columns, and other spare components of the instrument.

Monazite ID-TIMS U-Pb geochronology in the LAGIR laboratory, Rio de Janeiro State University: protocols and first applications to the assembly of Gondwana supercontinent in SE-Brazil

The chemical and spectrometric procedures of the U-Pb geochronology method on monazites, recently installed in the LAGIR laboratory, are described in detail. In addition, preliminary results on monazite samples from the Brasília and Ribeira belts are reported and discussed in the context of the regional geology. Several experiments for calibration of ion exchange chromatographic columns with the AG-1x8 resin, were performed with HCl, using dissolved natural monazite samples. The Pb blanks of reagents are ∼0.5 pg/g in acids and ∼1 pg/g in H2O. The total Pb blanks in chemical procedures were below 22 pg. Preliminary results are presented from three case studies related to Brasiliano orogenic belts of SE-Brazil, which correlate very well with previous age determinations from literature: two sub-concordant grains from an Araxá Group quartzite (southern Brasília belt) define a concordia age of 602.6 ±1.4 Ma; a -0.8% discordant grain from a quartzite of the São Fidelis Group (Costeiro Domain, central Ribeira belt) yielded a concordia age of 535.3 ± 2.4 Ma; two 0.4 % and 1.3 % discordant monazite grains from the post-collisional Itaoca Granite (Costeiro Domain, central Ribeira belt) define a concordia age of 476.4 ± 1.8 Ma.

Truths and myths about the determination of molar mass distribution of synthetic and natural polymers by size exclusion chromatography

ABSTRACTThis article discusses various aspects of the determination of molar mass distribution by means of size exclusion chromatography (SEC) in various application modes. The effects of erroneous specific refractive index increment (dn/dc), branching, column performance, and enthalpic interactions on the results obtained by different SEC techniques are discussed. Combination of SEC and a light scattering detector represents the most direct way to the molar mass distribution of all natural and synthetic polymers as it completely eliminates the need for column calibration and to a certain extent eliminates the dependence of the obtained results on some operational variables such as flow rate, temperature, or injected mass. A multiangle light scattering (MALS) photometer has become the most frequently used light scattering detector capable of determination of molecular size as another important polymer characteristic. This article contrasts SEC‐MALS method with other application modes of SEC from the viewpoint of some frequent confusions and misunderstandings. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 40111.

Now for my next trick: Absolute Molecular Weights by Column Calibration

Now for my next trick: Absolute Molecular Weights by Column Calibration

Now for my trick: Absolute Molecular Weights by Column Calibration

Now for my trick: Absolute Molecular Weights by Column Calibration

Improved predictions of saturated and unsaturated zone drawdowns in a heterogeneous unconfined aquifer via transient hydraulic tomography: Laboratory sandbox experiments

► Uniform parameters lead to biased drawdown predictions in the (un)saturated zones. ► Accounting for aquifer heterogeneity yields significant improvements in predictions. ► Considering unsaturated zone (UZ) heterogeneity produced a minor improvement. ► UZ heterogeneity plays a minor role in forming the S shape drawdown curve. ► Hydraulic tomography can be used to characterize unconfined aquifers. Interpretation of pumping tests in unconfined aquifers has largely been based on analytical solutions that disregard aquifer heterogeneity. In this study, we investigate whether the prediction of drawdown responses in a heterogeneous unconfined aquifer and the unsaturated zone above it with a variably saturated groundwater flow model can be improved by including information on hydraulic conductivity ( K ) and specific storage ( S s ) from transient hydraulic tomography (THT). We also investigate whether these predictions are affected by the use of unsaturated flow parameters estimated through laboratory hanging column experiments or calibration of in situ drainage curves. To investigate these issues, we designed and conducted laboratory sandbox experiments to characterize the saturated and unsaturated properties of a heterogeneous unconfined aquifer. Specifically, we conducted pumping tests under fully saturated conditions and interpreted the drawdown responses by treating the medium to be either homogeneous or heterogeneous. We then conducted another pumping test and allowed the water table to drop, similar to a pumping test in an unconfined aquifer. Simulations conducted using a variably saturated flow model revealed: (1) homogeneous parameters in the saturated and unsaturated zones have a difficult time predicting the responses of the heterogeneous unconfined aquifer; (2) heterogeneous saturated hydraulic parameter distributions obtained via THT yielded significantly improved drawdown predictions in the saturated zone of the unconfined aquifer; and (3) considering heterogeneity of unsaturated zone parameters produced a minor improvement in predictions in the unsaturated zone, but not the saturated zone. These results seem to support the finding by Mao et al. (2011) that spatial variability in the unsaturated zone plays a minor role in the formation of the S-shape drawdown-time curve observed during pumping in an unconfined aquifer.

“True” Concentration Determination Using a Concentration and Composition Sensitive Infrared Detector for Molecular Weight Characterization of Polyolefins

AbstractSummary: The infrared detector with a composition channel provides the ability to construct “true” concentration of the polyethylene‐polypropylene copolymer and polymer blends to compensate the different infrared responses to each component. Using this “true” concentration constructed by the infrared detector, triple detector gel permeation chromatography (GPC) could generate conventional (by column calibration) and absolute (by laser light scattering) molecular weight and molecular weight distribution data comparable to data obtained by using the concentration obtained by a differential reflex index concentration detector. In addition to its unique feature of in‐situ composition detection, the infrared detector in its mass detection mode possesses the advantages of faster equilibrium time, more stable baseline, less effect from temperature variation, and low sensitivity to moisture or air in the solvent.

Functional and Metabolic Effects of Adaptive Glycerol Kinase (GLPK) Mutants in Escherichia coli

Herein we measure the effect of four adaptive non-synonymous mutations to the glycerol kinase (glpK) gene on catalytic function and regulation, to identify changes that correlate to increased fitness in glycerol media. The mutations significantly reduce affinity for the allosteric inhibitor fructose-1,6-bisphosphate (FBP) and formation of the tetramer, which are structurally related, in a manner that correlates inversely with imparted fitness during growth on glycerol, which strongly suggests that these enzymatic parameters drive growth improvement. Counterintuitively, the glpK mutations also increase glycerol-induced auto-catabolite repression that reduces glpK transcription in a manner that correlates to fitness. This suggests that increased specific GlpK activity is attenuated by negative feedback on glpK expression via catabolite repression, possibly to prevent methylglyoxal toxicity. We additionally report that glpK mutations were fixed in 47 of 50 independent glycerol-adapted lineages. By far the most frequently mutated locus (nucleotide 218) was mutated in 20 lineages, strongly suggesting this position has an elevated mutation rate. This study demonstrates that fitness correlations can be used to interrogate adaptive processes at the protein level and to identify the regulatory constraints underlying selection and improved growth.

Development and qualification of a size exclusion chromatography coupled with multiangle light scattering method for molecular weight determination of unfractionated heparin

The molecular weight of unfractionated heparin was determined by size exclusion chromatography (SEC) coupled with multiangle light scattering (MALS) detection. The SEC/MALS method determines absolute molecular weight directly from the angular dependence of scattered light intensity as a function of concentration and does not rely on molecular weight standards for column calibration. The SEC/MALS method developed at Scientific Protein Laboratories was qualified in terms of specificity, precision, robustness, and accuracy. By eliminating the requirement of well-characterized molecular weight standards derived from heparin, the present procedure represents a clear improvement over the column calibration methods used in molecular weight determination. The SEC/MALS method is suitable for routine quality control of unfractionated heparin.

Identification of a Coiled Coil in Werner Syndrome Protein That Facilitates Multimerization and Promotes Exonuclease Processivity

Werner syndrome (WS) is a rare progeroid disorder characterized by genomic instability, increased cancer incidence, and early onset of a variety of aging pathologies. WS is unique among early aging syndromes in that affected individuals are developmentally normal, and phenotypic onset is in early adulthood. The protein defective in WS (WRN) is a member of the large RecQ family of helicases but is unique among this family in having an exonuclease. RecQ helicases form multimers, but the mechanism and consequence of multimerization remain incompletely defined. Here, we identify a novel heptad repeat coiled coil region between the WRN nuclease and helicase domains that facilitates multimerization of WRN. We mapped a novel and unique DNA-dependent protein kinase phosphorylation site proximal to the WRN multimerization region. However, phosphorylation at this site affected neither exonuclease activity nor multimeric state. We found that WRN nuclease is stimulated by DNA-dependent protein kinase independently of kinase activity or WRN nuclease multimeric status. In addition, WRN nuclease multimerization significantly increased nuclease processivity. We found that the novel WRN coiled coil domain is necessary for multimerization of the nuclease domain and sufficient to multimerize with full-length WRN in human cells. Importantly, correct homomultimerization is required for WRN function in vivo as overexpression of this multimerization domain caused increased sensitivity to camptothecin and 4-nitroquinoline 1-oxide similar to that in cells lacking functional WRN protein.

Human Plasma Copper Proteins Speciation by Size Exclusion Chromatography Coupled to Inductively Coupled Plasma Mass Spectrometry. Solutions for Columns Calibration by Sulfur Detection

Among the hyphenated techniques used to probe and identify metalloproteins, size exclusion chromatography coupled to inductively coupled plasma mass spectrometry (SEC-ICP-MS) has shown to have a central place. However, the calibration of SEC columns reveals to be tedious and always involves UV detection prior to ICP-MS. The presence of sulfur in 98% of proteins allows their detection by quadrupole ICP-MS, despite the isobaric interference ((16)O(16)O) on S, by monitoring (32)S(16)O at mass to charge ratio (m/z) 48. The formation of SO occurs spontaneously in the argon plasma but can be optimized by the introduction of oxygen gas into a reaction cell (RC) to achieve nM levels. In this article, sulfur detection was discussed upon instrumental conditions and S detection was then optimized by applying O(2) as a reaction gas. SO formation was used to calibrate SEC columns without UV detection. This simple SEC-ICP-MS method was used for plasma copper proteins in plasma healthy subjects (HS) and an untreated Wilson disease (WD) patient. Copper proteins identified in healthy subjects were transcuprein, ceruloplasmin (Cp) and albumin. The method led to results in good agreement with other methods of determination. Copper bound to Cp in the WD patient was lowered with regard to the HS, and the exchangeable Cu was highly increased.