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Summary: Human stem cell-derived models of development and neurodegenerative diseases are challenged by cellular immaturity in vitro. Microengineered organ-on-chip (or Organ-Chip) systems are designed to emulate microvolume cytoarchitecture and enable co-culture of distinct cell types. Brain microvascular endothelial cells (BMECs) share common signaling pathways with neurons early in development, but their contribution to human neuronal maturation is largely unknown. To study this interaction and influence of microculture, we derived both spinal motor neurons and BMECs from human induced pluripotent stem cells and observed increased calcium transient function and Chip-specific gene expression in Organ-Chips compared with 96-well plates. Seeding BMECs in the Organ-Chip led to vascular-neural interaction and specific gene activation that further enhanced neuronal function and in vivo-like signatures. The results show that the vascular system has specific maturation effects on spinal cord neural tissue, and the use of Organ-Chips can move stem cell models closer to an in vivo condition. : Sances et al. combine Organ-Chip technology with human induced pluripotent stem cell-derived spinal motor neurons to study the maturation effects of Organ-Chip culture. By including microvascular cells also derived from the same patient line, the authors show enhancement of neuronal function, reproduction of vascular-neuron pathways, and specific gene activation that resembles in vivo spinal cord development. Keywords: organ-on-chip, spinal cord, iPSC, disease modeling, amyotrophic lateral sclerosis, microphysiological system, brain microvascular endothelial cells, spinal motor neurons, vasculature, microfluidic device

Wnt signaling is intrinsic to mouse embryonic stem cell self-renewal. Therefore, it is surprising that reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) is not strongly enhanced by Wnt signaling. Here, we demonstrate that active Wnt signaling inhibits the early stage of reprogramming to iPSCs, whereas it is required and even stimulating during the late stage. Mechanistically, this biphasic effect of Wnt signaling is accompanied by a change in the requirement of all four of its transcriptional effectors: T cell factor 1 (Tcf1), Lef1, Tcf3, and Tcf4. For example, Tcf3 and Tcf4 are stimulatory early but inhibitory late in the reprogramming process. Accordingly, ectopic expression of Tcf3 early in reprogramming combined with its loss of function late enables efficient reprogramming in the absence of ectopic Sox2. Together, our data indicate that the stepwise process of reprogramming to iPSCs is critically dependent on the stage-specific control and action of all four Tcfs and Wnt signaling.

To determine the efficiency that can be obtained in a packed-bed liquid-chromatography column for a particular analyte, a correct determination of the molecular and effective diffusion coefficients (D m and D eff ) of the analyte is required. The latter is usually obtained via peak parking experiments wherein the flow is stopped. As a result, the column pressure rapidly dissipates and the measurement is essentially conducted at ambient pressure. This is problematic for analytes whose retention depends on pressure, such as proteins and potentially other large (dipolar) molecules. In that case, a conventional peak parking experiment is expected to lead to large errors in D eff . To obtain a better estimate ofD eff , the present study reports on the use of a set-up enabling peak parking measurements under pressurized conditions. This approach allowed us to report, for the first time, D eff for proteins at elevated pressure under retained conditions. First, D eff was determined at a (average) pressure of about 105 bar for a set of proteins with varying size, namely: bradykinin, insulin, lysozyme, β-lactoglobulin, and carbonic anhydrase in a column packed with 400 Å core-shell particles. The obtained data were then compared to those of several small analytes: acetophenone, propiophenone, benzophenone, valerophenone, and hexanophenone. A clear trend between D eff and analyte size was observed. The set-up was then used to determine D eff of bradykinin and lysozyme at variable (average) pressures ranging from 28 bar to 430 bar. These experiments showed a decrease in intra-particle and surface diffusion with pressure, which was larger for lysozyme than bradykinin. The data show that pressurized peak parking experiments are vital to correctly determine D eff when the analyte retention varies significantly with pressure., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Having fundamental insights in the properties of stationary phases and in the driving forces during the column packing process, is crucial to obtain highly efficient and robust packed-bed column technologies for use in separation science. Here we discuss the properties of the most commonly-used stationary-phases, i.e., silica particulate materials, including fully-porous and core-shell silica particles and provide an overview of the most commonly used column hardware and available frit technologies. The different packing approaches that are considered are dry packing, high-pressure slurry packing, electrokinetic packing, and packing using centrifugal forces. In particular, sedimentation of particles in slurries and particle interaction during the packing process affecting the resulting sphere packing are discussed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Dalbavancin is a lipoglycopeptide antibiotic used off-label to treat serious gram-positive infections, including infections secondary to methicillin-resistant Staphylococcus aureus (MRSA). Dalbavancin has unique pharmacokinetic parameters and has a role in therapy for treating vulnerable patients, including intravenous drug users, who have challenges complying with typical care plans for serious infections. While there is data indicating successful clinical use of dalbavancin in patients with history of intravenous drug use as well as pharmacokinetic-pharmacodynamic data assessing dalbavancin in obesity, there is a lack of information regarding clinical effects of dalbavancin in patients with extreme obesity, especially in patients with concomitant drug use. This case report describes a 40-year-old morbidly obese female actively using intravenous drugs who developed prolonged MRSA bacteremia without a recognizable focus. Despite partial treatment with dalbavancin, the patient developed osteomyelitis and discitis of the spine with associated epidural phlegmon, likely complications of the MRSA bacteremia., (Copyright © 2022 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Few studies have investigated the prevalence of polybrominated diphenyl ethers (PBDEs) in deep-sea sharks. In this study, the levels and profiles of PBDEs were determined in liver samples of eight different species of deep-sea sharks collected in Suruga Bay, Japan. Widespread contamination of PBDEs in the deep-sea environment was reconfirmed in this study as these persistent organic pollutants (POPs) were detected in all specimens analyzed. Mean ΣPBDE levels in the deep-sea sharks ranged from 7 to 517 ng/g of lipid weight. The distribution patterns of BDE homologues were similar in all species where tetra-BDEs provided the dominant contribution to total PBDEs (46%). PBDEs levels were similar to, or higher than, those seen in other deep-sea sharks from different regions. The levels of PBDEs were likely to reflect their feeding preferences as higher PBDE levels were seen in species with higher trophic positions., (Copyright © 2022. Published by Elsevier Ltd.)

Background: Body mass index (BMI) correlates with clinical outcomes in cystic fibrosis but has limitations.  Body composition aberrations in CF are multifactorial. We sought to evaluate body composition and relationships with pulmonary function, bone health, and hospital admissions. Other aims included defining body composition indices in a cohort of children with CF., Methods: We conducted a retrospective review of patients with CF, age 8-18 years, seen at Nationwide Children's Hospital (2015-2020). Indices of body composition measured by dual-energy x-ray absorptiometry(DXA) scans.  Data included fat mass, fat-free mass (FFM), forced expiratory volume in one second (FEV1%), bone mineral density (BMD), and hospital admissions. NWA was defined as BMI 5 th -85 th  percentile, body fat percentage >85 th  percentile. FFMD defined as FFMI <10 th  percentile., Statistics: T-tests compared NWA, FFMD and clinical measurements. Pearson correlations analyzed fat-free mass index (FFMI), fat mass index (FMI), BMI and clinical measurements., Results: This study included 114 patients.  Mean age 12 years, 72 female. A high prevalence of FFMD existed (n=66, 38.6%).  FMI and FFMI correlated with FEV1% (r: 0.23, p:0.01, r: 0.36, p<0.001, respectively) and BMD (r: 0.29, p:0.002). FMI and hospital admissions were related (r:-0.23,p:0.01). FFMD was associated with 9.5% lower FEV1% (p=0.001) and lower BMD Z-score by 1.1 (p<0.001) when compared to no FFMD., Conclusion: This cohort of children with CF had a high prevalence of FFMD and low prevalence of NWA.  FFMD was associated with worsened clinical measurements. Patients with FFMD need additional exercise or nutritional intervention. Heterogeneity of body composition definitions creates need for more research., Competing Interests: Declaration of Competing Interest Declarations of interest: none. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors., (Copyright © 2021 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

This article describes the use of a new prototype column hardware made with 1.5 mm internal diameter (i.d.) and demonstrates some benefits over the 1.0 mm i.d. micro-bore column. The performance of 2.1, 1.5 and 1.0 mm i.d. columns were systematically compared. With the 1.5 mm i.d. column, the loss of apparent column efficiency can be significantly reduced compared to 1.0 mm i.d. columns in both isocratic and gradient elution modes. In the end, the 1.5 mm i.d. column is almost comparable to 2.1 mm i.d. column from a peak broadening point of view. The advantages of the 1.5 mm i.d. hardware vs 2.1 mm i.d. narrow-bore columns are the lower sample and solvent consumption, and reduced frictional heating effects due to decreased operating flow rates., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier B.V.)

The abyss (3500-6500 m) covers the bulk of the deep ocean floor yet little is known about the extent of plastic debris on the abyssal seafloor. Using video imagery we undertook a quantitative assessment of the debris present on the abyssal seafloor (5700-5800 m depth) beneath the Kuroshio Extension current system in the Northwest Pacific. This body of water is one of the major transit pathways for the massive amounts of debris that are entering the North Pacific Ocean from Asia. Shallower sites (1400-1500 m depth) were also investigated for comparison. The dominant type of debris was single-use plastics - mainly bags and food packaging. The density of the plastic debris (mean 4561 items/km 2 ) in the abyssal zone was the highest recorded for an abyssal plain suggesting that the deep-sea basin in the Northwest Pacific is a significant reservoir of plastic debris., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The article describes the development of new stationary phases for the analysis of proteins in reversed phase liquid chromatography (RPLC). The goal was to have columns offering high recovery at low temperature, low hydrophobicity and novel selectivity. For this purpose, three different ligands bound onto the surface of superficially porous silica-based particles were compared, including trimethyl-silane (C1), ethyl-dimethyl-silane (C2) and N-(trifluoroacetomidyl)-propyl-diisopropylsilane (ES-LH). These three phases were compared with two commercial RPLC phases. In terms of protein recovery, the new ES-LH stationary phase clearly outperforms the other phases for any type of biopharmaceutical sample, and can already be successfully used at a temperature of only 60°C. In terms of retention, the new ES-LH and C1 materials were the less retentive ones, requiring lower organic solvent in the mobile phase. However, it is important to mention that the stability of C1 phase was critical under acidic, high temperature conditions. Finally, some differences were observed in terms of selectivity, particularly for the ES-LH column. Besides the chemical nature of the stationary phase, it was found that the nature of organic modifier also plays a key role in selectivity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

In this study, 31 racemates of Nα-FMOC (fluorenylmethoxycarbonyl) amino acids (AAs) with different chemico-physical characteristics (neutral nonpolar, neutral polar, acidic and basic) have been successfully resolved in fast enantioselective chromatography on recently-developed zwitterionic-teicoplanin chiral stationary phases (CSPs). The CSPs were prepared by covalently bonding the teicoplanin selector on fully-porous particles of narrow dispersion particle-size distribution (particle diameter 1.9 µm) and superficially-porous particles (2.0 µm). Both the zwitterionic-teicoplanin CSPs have proved to be ideal media for the separation of this important class of compounds. In particular, the zwitterionic CSP prepared on superficially-porous particles exhibited superior enantioselectivity and resolution, compared to that made of fully porous particles, in virtue of more favorable thermodynamics. The zwitterionic nature of these CSPs allowed avoiding the annoying effect of Donnan's exclusion of enantiomers from the stationary phase. This effect, on the opposite, was frequently observed on a commercial teicoplanin CSP (Teicoshell) employed for comparative purposes. Noticeably, on the zwitterionic-teicoplanin CSPs, by using either acetonitrile- or methanol-rich mobile phases (MPs), it was possible to favor speed over enantioresolution and vice versa. This work gives further replies to the request for rapid determination of enantiomeric excess of Nα-FMOC proteinogenic (and non-proteinogenic) AAs, typically used as preferred chiral synthons in the solid-phase synthesis of therapeutic peptides., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A novel column-coupling approach is suggested to improve both the selectivity and efficiency of protein separations in liquid chromatography. Protein separations often suffer from limited selectivity or not appropriate resolving power. For a new biopharmaceutical product, the identification of the main and minor variant species is required. For that purpose, often offline collection fractioning is applied which is time consuming and regularly dilute the samples to an unacceptable extent. By serially coupling columns in the order of their increasing retentivity and applying "multi-isocratic" elution mode, indeed any (arbitrary) selectivity can be attained. Moreover, if a protein peak is trapped at the inlet of a later column segment - of a coupled system -, its band will be refocused and elute in unprecedented sharp peak. Furthermore, it becomes possible to perform online on-column fractioning of protein species within a very short analysis time (∼ 1 min) and without sample dilution. Two-, three- or multiple column systems can be developed and applied for complex sample separations (such as antibody mixtures). This new methodology can be particularly useful to improve the analysis (and therefore, safety and quality) of therapeutic mAbs and related products and offers benefits compared to offline fractionating. It is also demonstrated in this proof of concept study, that methyl (C1) modified RP phase has a great potential for protein separations despite it is not commercially available in state-of-the-art wide pore superficially porous particle format.., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The performance of curtain flow chromatography column technology with MS detection was evaluated for the analysis of labile compounds. The curtain flow column design allows for separations that are faster and/or more sensitive than conventional columns, depending on how exactly the curtain flow column is configured. For example, when mass spectral detection is employed, the curtain flow column can yield separations that are 5-times faster than conventional columns when the curtain flow and the conventional columns have the same internal diameter. Or when the internal diameter of the conventional column is reduced in order to yield the same analytical through-put as the curtain flow column, the sensitivity on the curtain flow column can be as much as 66-fold higher than the conventional column. As a consequence of the higher analytical through-put less standardization is required in the analysis of labile compounds because less sample degradation is apparent. Consequently the sample integrity is preserved yielding data of a higher quality., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Analytical scale active flow technology first generation silica monolithic columns kitted out in curtain flow mode of operation were studied for the first time. A series of tests were undertaken assessing the column efficiency, peak asymmetry and detection sensitivity. Two curtain flow columns were tested, one with a fixed outlet ratio of 10% through the central exit port, the other with 30%. Tests were carried out using a wide range in inlet flow segmentation ratios. The performance of the curtain flow columns were compared to a conventional monolithic column. The gain in theoretical plates achieved in the curtain flow mode of operation was as much as 130%, with almost Gaussian bands being obtained. Detection sensitivity increased by as much as 250% under optimal detection conditions. The permeability advantage of the monolithic structure together with the active flow technology makes it a priceless tool for high throughput, sensitive, low detection volume analyses., (Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.)

The increase of the operating pressure in Liquid Chromatography, has been one of the crucial steps toward faster and more efficient separations. In the present contribution, it was investigated if the pressure limits for narrow-bore columns (2.1mm ID) could be increased beyond those of commercially available (1300bar) instrumentation without performance loss. Whereas previous studies applying pressures higher than 2000bar were limited to the use of columns with a diameter smaller or equal to 1mm, it is a difficult feat to expand this to 2.1mm ID given that viscous-heating effects increase according to the fifth power of the column radius. A prototype LC set-up was realized, allowing to operate at pressures up to 2600bar (260MPa) for large separation volumes (>5mL). The performance of an in-house-built injector was compared at 800bar to commercially available injectors, yielding equal performance but twice the maximum pressure rating. The performance of (coupled) custom columns packed with fully porous and superficially porous particles were assessed at ultra-high-pressure conditions. Increasing the inlet pressure from 800 to 2400bar and scaling the column length proportionally (from 150mm to 450mm), resulted in the theoretically expected linear increase in plate count from 20,000 to 59,000. A maximum plate number of 81,000 was realized using a 600mm long (coupled) column at 2600bar. Viscous-heating effects were diminished by insulating coupled columns and applying an intermediate-cooling strategy in a forced-air oven., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The variation in mobile phase velocity as a function of the column radius has been shown to be a major limitation in the efficiency of HPLC columns. One contributing factor to the variability in the flow velocity stems from the heterogeneity in the radial packing density, leading to what has been described as the 'wall-effect'. The wall-effect generates parabolic-type elution profiles, which dilutes the sample and creates tailing bands. In this communication a new column technology is discussed that has been designed to overcome the wall effect, minimising the limitations associated with packing heterogeneity. This technology has been referred to as active flow technology and consists of two types of column designs, parallel segmented flow and curtain flow. In both these column designs sample that elutes through the column in the radial central region of the bed is separated from the flow that elutes along the wall region. Hence, the sample that elutes through the most efficiently packed region of the bed is collected to the detector. As a consequence more theoretical plates are obtained, and sensitivity is increased since the sample is not diluted by the diffuse tail. Sensitivity is enhanced further in the curtain flow design. The benefits of these new columns are discussed., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Active flow technology (AFT) columns are designed to minimise inefficient flow processes associated with the column wall and radial heterogeneity of the stationary phase bed. This study is the first to investigate AFT on an analytical scale 4.6mm internal diameter first-generation silica monolith. The performance was compared to a conventional first-generation silica monolith and it was observed that the AFT monolith had an increase in efficiency values that ranged from 15 to 111%; the trend demonstrating efficiency gains increasing as the volumetric flow to the detector was decreased, but with no loss in sensitivity., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A preliminary investigation was undertaken to assess the performance of a new chromatography column technology in applications involving liquid chromatography coupled to mass spectrometry. The new column design allows mobile phase and solute to be extracted from the radial central region of the column, which reduces the solvent load to the mass spectrometer and improves separation efficiency. Effectively the column functions as a 'wall-less' column. The advantages of this design is that the analysis through-put can be increased by a factor of five, while at the same time there is a reduction in baseline noise, which results in an increase in the signal to noise response by up to 10-fold in comparison to standard columns with the same internal diameter and approaching 66-fold in comparison to standard columns with the same virtual internal diameter., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The analysis of antioxidants from complex samples is conveniently achieved using liquid chromatography, which provides sample fraction, coupled with an on-line antioxidant assay, which provides detection. One particularly useful on-line antioxidant assay that has routinely been coupled with HPLC involves the diphenylpicrylhydrazyl radical (DPPH), which provides a positive test for phenolic antioxidants through a decolorisation of the DPPH reagent. A limitation of this assay, however, is the need to employ a reaction coil, which is often large with respect to the peak volume, consequently adding substantial band broadening to the separation. In this study we introduce a new concept that can be employed for systems requiring post column derivatisations, such as the DPPH assay. We have termed this 'reaction flow' chromatography, whereby, the derivatisation reagent can be added directly into one of the outlet ports of a parallel segmented flow column. Subsequently, the mixing between the derivatising reagent and the solute is very efficient removing the need to employ reaction coils. The concept is tested here using the DPPH assay for the analysis of antioxidants in samples derived from natural origin., (Copyright © 2013 Elsevier B.V. All rights reserved.)

There is a continuing challenge in improving the separation speed while keeping both the resolution high and the back pressure low in high performance liquid chromatography (HPLC). The recent renaissance of core-shell (or fused-core) silica particles has shown great promise in this respect. However, the fused-core silica particles are typically synthesized by the time-consuming multiple-step layer-by-layer technique. An one-pot synthesis of spheres-on-sphere (SOS) silica microspheres is presented here. The preparation parameters including Si precursors, mixing methods (magnetic stirring, mechanical stirring and homogenization), heating (microwave heating and conventional heating), and reaction temperature are investigated in order to control the morphology and improve the size distribution of the SOS particles. The improvement and influence on SOS morphology, particle size and particle size distribution are discussed. Furthermore, the calcined and modified SOS particles are packed into stainless steel columns, which are then assessed for the separation of various test mixtures containing small molecules and proteins under normal phase, reversed phase, and HILIC conditions. The HPLC tests demonstrate fast and efficient separation with very low back pressure, suggesting that SOS particles are a type of new and highly promising packing materials for liquid chromatography., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A new column format known as parallel segmented flow has recently been introduced, whereby improvements in column performance are observed. These improvements are achieved via the separation of eluent from the column core from that of the column wall region. The segmentation of flow is accomplished immediately as the eluent exits the column through the use of a multi-channel end fitting. The ratio of flow exiting through the column central port relative to the peripheral ports, known as the segmentation ratio, can be tuned to optimise chromatographic performance. Investigations into the use of parallel segmented flow chromatography columns have demonstrated increased sensitivity and theoretical plates in analytical scale isocratic separations, but so far no studies have detailed the performance of these columns in gradient elution. The current study addresses the performance of parallel segmented flow columns in gradient elution, detailing the reproducibility of the gradient at various segmentation ratios and compares the performance to conventional columns. The study found that there was no observable difference in the gradient shape, or reproducibility of the gradient profiles generated at any segmentation ratio, tested on three different types of stationary phases. A separation of an 11-component test mixture verified that the primary advantage of parallel segmented flow columns was that the peak volume was reduced in proportion to the segmentation ratio., (Copyright © 2012. Published by Elsevier B.V.)

This paper reports on the thermodynamic and kinetic evaluation of a new ultra-high performance liquid chromatography broad-spectrum Pirkle-type chiral stationary phase (CSP) for enantioselective applications (eUHPLC). The well-known Whelk-O1 selector was covalently immobilized onto 1.7-μm high-surface-area, porous spherical silica particles to produce a totally synthetic, covalently bonded CSP that was packed into 150 mm, 100mm, 75 mm and 50mm columns, either 4.6 or 3.0mm ID. A 100 mm × 4.6 mm ID column was fully characterized from a kinetic and thermodynamic point of view, using as reference a conventional HPLC Whelk-O1 column, 250 mm×4.6mm ID, based on 5-μm porous silica particles. On the eUHPLC column, van Deemter plots generated H(min) values of 3.53 μm for 1,3-dinitrobenzene, at an interstitial mobile phase linear velocity (μ(inter)) of 5.07 mm/s, and H(min) of 4.26 and 4.17 μm for the two enantiomers of acenaphthenol, at μ(inter) of 4.85 mm/s and 4.24 mm/s, respectively. Resolution of 21 enantiomeric pairs including alcohols, epoxides, sulfoxides, phosphine oxides, benzodiazepines and 2-aryloxyproprionic esters used as herbicides, were obtained with significant advantages in terms of efficiency and analysis time. Speed gain factors were calculated for the different column geometries (150 mm, 100mm, 75 mm and 50mm, either 4.6 or 3.0mm ID), with respect to the standard HPLC column (250 mm ×4.6 mm ID), and were as high as 13, in the case of the 50-mm-long column, affording sub-minute separations of enantiomers with excellent resolution factors. In particular, trans-stilbene oxide was resolved in only 10s, while a 50 mm×3.0 mm ID column was used as a compromise between reduced mobile phase consumption (less than 1 mL per analysis) and smaller extra-column band-broadening effect. Given the relatively low viscosity in NP mode, and the excellent permeability of these eUHPLC columns, with backpressure values under 600 bar for a wide range of flow rates, the use of standard HPLC hardware is possible. In this case, however, a significant loss in resolution is observed, compared to the UHPLC instrumentation, if no modifications are introduced in the HPLC apparatus to reduce extra-column variance. The excellent efficiency and selectivity, conjugated with the very high-throughput and the ultra-fast analysis time, prove the potentials of the eUHPLC Whelk-O1 columns in the development of enantioselective UHPLC methods., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Narrow bore columns find advantage in HPLC applications when volumetric flow is important, For example, for detection processes that are volume limited. Yet there are significant drawbacks to narrow bore columns. Due to their small column volume relative to analytical scale columns, narrow bore columns are more affected by system dead volume. In addition the wall effect and the variation in packing density from the centre to the wall are more significant in these columns relative to larger scale analytical columns. In this study we operate a 4.6mm i.d. parallel segmented flow column in such a manner that it emulates 2.1mm i.d. and 3.0mm i.d. columns. By using a parallel segmented flow column in this way, it was possible to combine the benefits of narrow bore and analytical scale columns., (Copyright © 2012 Elsevier B.V. All rights reserved.)

We present the first investigation into the utility of porous graphitic carbon (PGC) as a stationary phase in proteomic workflows involving complex samples. PGC offers chemical and physical robustness and is capable of withstanding extremes of pH and higher temperatures than traditional stationary phases, without the likelihood of catastrophic failure. In addition, unlike separations driven by ion exchange mechanisms, there is no requirement for high levels of non-volatile salts such as potassium chloride in the elution buffers, which must be removed prior to LC-MS analysis. Here we present data which demonstrate that PGC affords excellent peptide separation in a complex whole cell lysate digest sample, with good orthogonality to a typical low pH reversed-phase system. As strong cation exchange (SCX) is currently the most popular first dimension for 2D peptide separations, we chose to compare the performance of a PGC and SCX separation as the first dimension in a comprehensive 2D-LC-MS/MS workflow. A significant increase, in the region of 40%, in peptide identifications is reported with off-line PGC fractionation compared to SCX. Around 14,000 unique peptides were identified at an estimated false discovery rate of 1% (n=3 replicates) from starting material constituting only 100 μg of protein extract., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The effect of the addition of 25%, 50% and 75% (weight percent, wt%) of larger particles (resp. 3 and 5 μm) to a commercial batch of 1.9 μm particles has been investigated as an academic exercise to study the effects of particle size distribution on the kinetic performance of packed bed columns in a magnified way. Comparing the performance of the different mixtures in a kinetic plot, it could be irrefutably shown that the addition of larger particles to a commercial batch of small particles cannot be expected to lead to an improved kinetic performance. Whereas the addition of 25 wt% of larger particles still only has a minor negative effect, a significantly deteriorated performance is obtained when 50 or 75 wt% of larger particles are added. In this case, separation impedance number increases up to 200% were observed. Studying the packing structure through computational packing simulations, together with the experimental determination of the external porosity, helped in understanding the obtained results. This showed that small particles tend to settle in the flow-through pores surrounding the larger particles, leading to very high packing densities (external porosities as low as 32% were observed) and also negatively influencing the column permeability as well as the band broadening (because of the broadened flow-through pore size range)., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We report the first development of a novel, planar, microfluidic, graphitic carbon separations column utilizing an array of graphitic micropillars of diamond cross-section as the chromatographic stationary phase. 795 nm femtosecond laser ablation was employed to subtractively machine fluidic architectures and a micropillared array in a planar, graphitic substrate as a monolithic structure. A sample injector was integrated on-chip, together with fluid-flow distribution architectures to minimize band-broadening and ensure sample equi-distribution across the micro-pillared column width. The separations chip was interfaced directly to the ESI probe of a Thermofisher Surveyor mass spectrometer, enabling the detection of test-mixture analytes following their differential retention on the micro-pillared graphitic column, thus demonstrating the exciting potential of this novel separations format. Importantly, unlike porous, graphitic microspheres, the temperature and pressure resilience of the microfluidic device potentially enables use in subcritical H(2)O chromatography., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Three brush-type chiral stationary phases (CSPs) differing in the particle size of the starting silica particles have been prepared by covalent grafting of the pi-acidic bis-(3,5-dinitrobenzoyl)-derivative of trans-1,2-diaminocyclohexane (DACH-DNB). Starting silica particles of 4.3, 2.6 and 1.9micron were used to generate the final CSPs using an improved, highly reproducible synthetic methodology, that allowed to assemble and surface-graft the whole chiral selector in only two steps. The different CSPs have been packed in columns of various length and diameters, and fully characterized in terms of flow permeability, kinetic performances and enantioselectivity using a set of test solutes. Very high speed and high resolution applications together with stereodynamic HPLC examples are demonstrated on the columns with reduced particle diameters, on which separations of several enantiomeric pairs are routinely obtained with analysis times in the 15-40s range., (Copyright 2009 Elsevier B.V. All rights reserved.)

To study the influence of the particle size distribution (PSD), we measured the chromatographic performance of a series of sub-2 microm particle high performance liquid chromatography (HPLC) columns packed with four different particle mixtures having a purposely imposed different size distribution. Using the reduced kinetic plot representation by plotting the separation impedance (E(0)) versus the plate number ratio (N(opt)/N), the different columns could be classified according to their chromatographic performance without the need to specify a mean particle diameter or a molecular diffusion coefficient, as is needed in the classical reduced plate height and flow resistance analysis. The present analysis shows that it is not so much the width or span of the particle size distribution, but rather the presence of fines that greatly determines the chromatographic performance of particulate columns.

A series of studies has been carried out on the effect of refluxing silica chromatography particles for 0.5 h and 18 h in water, dilute hydrochloric acid and dilute hydrofluoric acid. The bulk and surface trace metal concentrations were measured by inductively-coupled plasma atomic emission spectroscopy, static secondary ion mass spectrometry (SSIMS) and X-ray photoelectron spectroscopy. Diffuse reflectance Fourier transform infrared spectroscopy was used to determine changes in 'isolated" and "bonded" silanol groups. The chromatographic behaviour of a series of weakly basic analytes was investigated on C8 and C18 bonded phases manufactured from the acid-treated silicas. The different reflux treatments all resulted in a reduction in the numbers of isolated silanols compared with the untreated silica and SSIMS analysis suggested that the HF-treated silicas had undergone a more efficient surface rehydroxylation. Bulk trace metals were removed most effectively by the HF treatment, with the multivalent elements (Ti and Al) being the most difficult to remove. Surface specific analysis suggested that trace metals were removed more rapidly from the surface of the silica compared to the bulk matrix and that the acid treatments resulted in halide contamination of the silica surface. Evidence is presented to suggest that the bulk metal content of the silica is not representative of the concentration of metals at the chromatographic surface. The chromatographic investigations showed that the HF-treated silica gave substantially better performance towards weak bases than the HCl-treated silicas.

We previously reported that the cDNA for dentin phosphophoryn, a non-collagenous protein involved in dentin mineralization, was located immediately downstream from the 3' end of dentin sialoprotein, another functionally related non-collagenous dentin protein (Ritchie, H. H., and Wang, L-H. (1996) J. Biol. Chem. 269, 3689-3702). We now demonstrate unequivocally that at the genomic level in mammal, these two functionally related proteins are indeed transcribed as one transcriptional unit suggesting that their combined presence at specific mineralization sites in tooth germ is mandatory if dentin mineralization is to proceed.

Fibrin deposition is characteristic of inflammatory diseases. The monocytes is central to the inflammatory response and can affect fibrinolysis by expression of urokinase (u-PA) and plasminogen activator inhibitor types 1 and 2 (PAI-1 and PAI-2, respectively). This study examines whether thrombin, which promotes fibrin deposition, can contribute to fibrin persistence by modulating expression of proteins of the fibrinolytic system. Monocytes were isolated from human peripheral blood and analyzed for PAI-2, PAI-1, and u-PA antigens by enzyme-linked immunosorbent assay (ELISA). Monocytes responded to thrombin by increased expression of PAI-2 in a dose- and time-dependent manner, with maximal synthesis at a concentration of 1 U/mL to 10 U/mL. This trend was also evident for PAI-1, which was present at much lower levels. Thrombin and lipopolysaccharide (LPS) stimulated comparable levels of PAI-2, studied at the antigen and mRNA level. The dose effet of LPS on PAI-2 and PAI-1 was found to differ from that of thrombin. The level of u-PA was undetectable by ELISA and zymography in all samples. Thrombin stimulates PAI-2 synthesis by human monocytes, therefore creating an imbalance in the fibrinolytic system. This may contribute to persistence of fibrin, deposited during inflammation.

Two experiments were conducted to determine whether treatments with gonadotropin releasing hormone (GnRH) during the early postpartum period in suckled cows would induce ovulation and initiate regular estrous cycles. In Experiment I, 0, 100 or 200 microg of GnRH was given to 22 suckled Angus x Holstein cows at three and again at five weeks postpartum. Serum luteinizing hormone (LH) responses did not differ between cows given 100 or 200 microg of GnRH. Treatment with GnRH tended to increase the percentage of cows exhibiting estrus by 30 and 60 days postpartum, but reproductive performance during the breeding season did not differ among groups. In Experiment II, 70 suckled Hereford cows were given either no treatment or 200 microg of GnRH at 7 weeks postpartum. Cows given GnRH received either no treatment prior to GnRH or were separated from their calves for 24 hr prior to GnRH treatment. Half of the cows that were separated from their calves also received progesterone via a progesterone intravaginal device (PRID) for 12 days prior to calf removal. Treatment with GnRH alone tended to increase the percentage of anestrous cows which ovulated by 8 days after treatment. Calf removal did not increase the ovulatory response to GnRH, but PRID treatment did. More estrous periods were detected in GnRH-treated cows than in control cows during 20 days after GnRH treatment.