Characterization of hybrid organo-silica monoliths for possible application in the gradient elution of peptides.

We characterized thermally polymerized organo-silica hybrid monolithic capillaries to test their applicability in the gradient elution of peptides. We have used a single-pot approach utilizing 3-(methacryloyloxy)propyltrimethoxysilane (MPTMS), ethylene dimethacrylate (EDMA), and n-octadecyl methacrylate (ODM) as functional monomers. The organo-silica monolith containing MPTMS and EDMA was compared with the stationary phase prepared by adding ODM to the original polymerization mixture. Column prepared using a three-monomer system provided a lower accessible volume of flow-through pores, a higher proportion of mesopores, and higher efficiency. We utilized isocratic and gradient elution data to predict peak widths in gradient elution. Both protocols provided comparable results and can be used for peptide peak width prediction. However, applying gradient elution data for peak width prediction seems simpler. Finally, we tested the effect of gradient time on achievable peak capacity in the gradient elution of peptides with a column prepared with a three-monomer system providing a higher peak capacity. However, the performance of hybrid organo-silica monolithic stationary phases in gradient elution of peptides must be improved compared to other monolithic stationary phases. The limiting factor is column efficiency in highly aqueous mobile phases, which needs to be focused on.

Evaluation of Mechanical Properties and Filler Interaction in the Field of SLA Polymeric Additive Manufacturing.

The paper deals with research focused on the use of fillers in the field of polymeric materials produced by additive technology SLA (stereolithography). The aim of the research is to evaluate 3D printing parameters, the mechanical properties (tensile strength, hardness), and the interaction of individual phases (polymer matrix and filler) in composite materials using SEM analysis. The tested fillers were cotton flakes and ground carbon fibres in different proportions. For the photosensitive resins, the use of cotton flakes as filler was found to have a positive effect on the mechanical properties not only under static but also under cyclic loading, which is a common cause of material failure in practice. The cyclic stress reference value was set at an amplitude of 5-50% of the maximum force required to break the pure resin in a static tensile test. A positive effect of fillers on the cyclic stress life of materials was demonstrated. The service life of pure resin was only 168 ± 29 cycles. The service life of materials with fillers increased to approximately 400 to 540 cycles for carbon fibre-based fillers and nearly 1000 cycles for cotton flake-based fillers, respectively. In this paper, new composite materials suitable for the use of SLA additive manufacturing techniques are presented. Research demonstrated the possibilities of adding cotton-based fillers in low-cost, commercially available resins. Furthermore, the importance of material research under cyclic loading was demonstrated.

Recent stationary phase-based fractionation strategies in proteomic analysis.

In this article, we review stationary phase-assisted sample prefractionation in proteomic analysis in 2019-2022 period. Applications are grouped according to the mode of retention used in the prefractionation step prior to the final analysis by low-pH reversed-phase liquid chromatography-tandem mass spectrometry. Online or offline instrumental configurations are also commented on, with a highlight on novel online platforms. From the articles reviewed in this period, the order of popularity of chromatographic modes for sample fractionation is affinity chromatography > size exclusion chromatography > hydrophilic interaction chromatography > high-pH reversed-phase liquid chromatography > ion exchange chromatography.

Closed reduction and minimally invasive screw osteosynthesis of Pipkin femoral head fractures.

BACKGROUND: Femoral posterior hip dislocation with associated femoral head fractures (Pipkin fractures) are rare high-energy injuries. Published treatment modalities involve conservative treatment, head fragment resection, open reduction and internal fixation, and total hip replacement. The experience with mini-invasive screw osteosynthesis of these fractures is the main focus of our study. METHODS: Seven Pipkin fractures (five Pipkin II and two Pipkin I) in six patients were treated by closed reduction of hip dislocation, followed by minimal invasive lag screw osteosynthesis. Cancellous screw(s) were inserted from the incision on the lateral hip through the femoral neck to the reduced fracture fragment. In all patients, postoperative CT was performed to check the quality of surgery. Active physiotherapy with immediate toe-touch weight bearing was the routine postoperative protocol. In all patients, radiological and clinical results were evaluated with the Thompson Epstein, Merle d'Aubigne and Postel score, and Harris hip score. RESULTS: All fractures united, and all femoral heads survived. Infectious complications were not observed, and no secondary surgery was needed. After an average follow-up of 18.4 months, the average Merle d'Aubigne and Postel score was 17.7 points, while the mean Harris hip score reached 98.1 points. The majority of patients achieved an excellent Thompson-Epstein clinical and radiological outcome. All patients returned to their original occupation. CONCLUSIONS: Mini-invasive screw osteosynthesis can be used for the treatment of Pipkin type I-II femoral head fractures. Successful reduction of hip dislocation and head fracture is necessary for using this technique. Long-term follow-up is necessary to confirm this technique.

Segmented two-dimensional liquid chromatography. Proof of concept study.

The separation in liquid chromatography is defined either by the space domain where it proceeds until the least retained analyte reaches the outlet of the column or by the time when individual analytes elute out of the column. These two approaches lead to the four possible combinations of two-dimensional liquid chromatography with online space x time coupling being the least experimentally feasible. Here, we show the development of a novel two-dimensional liquid chromatography method combining separation defined by space and the conventional elution-based separation. First-dimension column consisted of four capillary segments coupled serially via two-position six-port valves allowing an online and comprehensive transfer of analytes from the first to the second dimension. After initial experiments using homemade monolithic capillary columns, we tested commercially available columns in both dimensions. We ended with the combination of packed capillary columns in the first dimension and monolithic capillary column in the second dimension. We used a reversed-phase retention mechanism in the first spatial dimension, while HILIC was in the second, time-based dimension. We also developed a theoretical model to describe the proposed two-dimensional separation that was further confirmed by utilizing both an isocratic and gradient elution in the second dimension. Finally, we applied our experimental setup to separate neurotransmitters contained in human urine.

Reversed-Phase Liquid Chromatography of Peptides for Bottom-Up Proteomics: A Tutorial.

The performance of the current bottom-up liquid chromatography hyphenated with mass spectrometry (LC-MS) analyses has undoubtedly been fueled by spectacular progress in mass spectrometry. It is thus not surprising that the MS instrument attracts the most attention during LC-MS method development, whereas optimizing conditions for peptide separation using reversed-phase liquid chromatography (RPLC) remains somewhat in its shadow. Consequently, the wisdom of the fundaments of chromatography is slowly vanishing from some laboratories. However, the full potential of advanced MS instruments cannot be achieved without highly efficient RPLC. This is impossible to attain without understanding fundamental processes in the chromatographic system and the properties of peptides important for their chromatographic behavior. We wrote this tutorial intending to give practitioners an overview of critical aspects of peptide separation using RPLC to facilitate setting the LC parameters so that they can leverage the full capabilities of their MS instruments. After briefly introducing the gradient separation of peptides, we discuss their properties that affect the quality of LC-MS chromatograms the most. Next, we address the in-column and extra-column broadening. The last section is devoted to key parameters of LC-MS methods. We also extracted trends in practice from recent bottom-up proteomics studies and correlated them with the current knowledge on peptide RPLC separation.

Liquid chromatography at the University of Pardubice: A tribute to Professor Pavel Jandera.

Pavel Jandera was a world-leading analytical chemist who devoted his entire professional life to research in the field of high-performance liquid chromatography. During his scientific career, he worked at the Department of Analytical Chemistry at the University of Pardubice, Czech Republic. His greatest contribution to the field of liquid chromatography was the introduction of a comprehensive theory of liquid chromatography with programmed elution conditions. He was also involved in the research of gradient elution techniques in preparative chromatography, modeling of retention and selectivity in various phase systems, preparation of organic monolithic microcolumns, and, last but not least, in the development of theory and practical applications of two-dimensional liquid chromatography, mainly in the comprehensive form. In this review article, we have tried to capture the highlights of his scientific career and provide the readers with a detailed overview of Pavel Jandera's contribution to the evolution of separation sciences.

Retention prediction of monoamine neurotransmitters in gradient liquid chromatography.

Retention prediction of monoamine neurotransmitters has been compared for the generally applied linear solvent-strength model and quadratic polynomial three-parameter model. The design of experiments protocol has been applied to plan linear gradients within the experimental space with altered gradient time, mobile phase flow rate, and column temperature. Relative prediction errors increased at elevated temperature, which is more significant for the linear solvent-strength model when compared to the polynomial model. On the other hand, the predefined design of experiments space controls the retention time errors, as predictions for LC conditions that are outside of the plan are much less accurate and should be avoided. The final part of the work deals with the effect of extracolumn band dispersion on the peak capacity of linear gradients at various gradient times, mobile phase flow rates, and column temperature. The peak capacity determined for corrected experimental data were consistent with the published results dealing with the optimization of peak capacity in gradient elution.

A review on recent trends in the phosphoproteomics workflow. From sample preparation to data analysis.

Phosphorylation is one of the quickest post-translational modifications that controls downstream signaling pathways regulating processes like cell proliferation, survival, and differentiation. Nowadays, mass spectrometry-based phosphoproteomics is a well-established method providing unprecedented characterization and quantification of phosphorylated proteins and peptides in complex samples. A comprehensive phosphoproteomics workflow consists of protein digestion, phosphopeptide enrichment, sample fractionation, chromatographic separation, and final detection by mass spectrometry. Each of these stages provides its own contribution to overall data variability and should be optimized thoroughly. This review aims to provide an overview of current developments in individual steps of phosphoproteomics workflow with a special focus on applied analytical methods. Recent efforts in all experimental steps are discussed. Finally, possible future development in the field of (phospho)proteomics is proposed.

Strategy of pulseless pink supracondylar humerus fracture treatment in children: a comparison of two approaches.

PURPOSE: The appropriate treatment of pulseless pink supracondylar humerus fractures (SCHF) remains controversial. In this study, the outcomes of two treatment approaches (with and without vascular surgery) were compared. MATERIAL AND METHODS: This was a retrospective multicenter study of patients with pulseless pink SCHFs treated in ten pediatric surgery, trauma, or orthopedics departments in the Czech and Slovak Republic between 2014 and 2018. RESULTS: Of the total 3608 cases of displaced SCHF, 125 had the pulseless pink SCHF. Of those, 91% (114/125) did not undergo vascular surgery and 9% (11/125) underwent vascular surgery. The patients who did undergo vascular surgery had radial artery pulsation restored more frequently in the operating room (73% vs. 36%; p = 0.02), within 6 h (91% vs. 45%; p = 0.004), and within 24 h of surgery (91% vs. 57%; p = 0.05). However, 72 h after surgery, there was no significant difference in palpable radial artery pulsation between the vascular surgery and the non-vascular surgery groups (91% vs. 74%; p = 0.24). Additionally, no significant differences in long-term neurological (9% vs. 22%; p = 0.46) or circulatory (9% vs. 7%; p = 0.57) deficits were found between the two groups. CONCLUSION: While vascular surgery in patients with pulseless pink SCHFs is associated with a more prompt restoration of radial artery pulsation, no statistical significant differences in terms of the restoration of neurological deficits or the risks of long-term neurological or circulatory deficits were found between patients with and without vascular surgery.

Current trends in the development of polymer-based monolithic stationary phases.

This review focuses on the development and applications of organic polymer monoliths, with special attention to the literature published in 2021. The latest protocols in the preparation of polymer monoliths are discussed. In particular, tailored surface modification using nanomaterials, the development of chiral stationary phases and development of stationary phases for capillary electrochromatography are reviewed. Furthermore, the optimization of pore forming solvents composition is also discussed. Finally, the use of monolithic stationary phases in sample treatment using solid-phase extraction and enrichment methods, molecularly imprinted polymers and enzymatic reactors is mentioned.

New Target for Precision Medicine Treatment of Giant-Cell Tumor of Bone: Sunitinib Is Effective in the Treatment of Neoplastic Stromal Cells with Activated PDGFRß Signaling.

Giant-cell tumor of bone (GCTB) is an intermediate type of primary bone tumor characterized by locally aggressive growth with metastatic potential. The aim of this study was to identify new druggable targets among the cell signaling molecules involved in GCTB tumorigenesis. Profiles of activated signaling proteins in fresh-frozen tumor samples and tumor-derived cell lines were determined using phosphoprotein arrays. Analysis of the obtained data revealed epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor beta (PDGFRß) as potential targets, but only the PDGFR inhibitor sunitinib caused a considerable decrease in stromal cell viability in vitro. Furthermore, in the case of a 17-year-old patient suffering from GCTB, we showed that the addition of sunitinib to the standard treatment of GCTB with the monoclonal antibody denosumab resulted in the complete depletion of multinucleated giant cells and mononuclear stromal cells in the tumor tissue. To summarize, the obtained data showed that a specific receptor tyrosine kinase (RTK) signaling pattern is activated in GCTB cells and plays an important role in the regulation of cell proliferation. Thus, activated RTKs and their downstream signaling pathways represent useful targets for precision treatment with low-molecular-weight inhibitors or with other types of modern biological therapy.

Photografting of polymer monoliths by a crosslinking monomer.

The surface of fifteen polymethacrylate monolithic stationary phases has been modified by a post-polymerization UV-initiated grafting reaction with bifunctional poly(ethylene glycol)dimethacrylate monomers. An effect of crosslinking monomer length, its concentration in the modification mixture, and a time of the modification reaction have been selected to control the extent of modification by a design of experiments protocol. Hydrodynamic and kinetic properties of prepared columns were characterized by capillary liquid chromatography. Regression analysis of determined data revealed that there is only a minor effect of modification reaction on column permeability, as it is rather controlled by the composition of the polymerization mixture used to prepare generic monolith. On the other hand, the utilization of shorter crosslinking monomer increased the formation of small pores and minimized mass transfer resistance effect. Both column efficiency and mass transfer resistance also improved when a lower concentration of crosslinking monomer in the modification mixture was used. Photografting modification decreased a negative effect of mass transfer resistance related to a crosslink density gradient and allowed fast isocratic separations of dopamine metabolism-related compounds. Developed preparation protocol might be further utilized in the preparation of monolithic stationary phases in microfluidic devices.

Dual-retention mechanism of dopamine-related compounds on monolithic stationary phase with zwitterion functionality.

Seven retention models have been selected to describe a dual-retention behavior of ten dopamine-related compounds on polymer-based monolithic stationary phase with zwitterion sulfobetaine functionality. Regression quality, as well as a statistical significance of individual regression parameters, have been evaluated. Better regression performance showed two four-parameter models when compared to three-parameter models. On the other hand, limited number of experimental points disqualified statistical robustness of four-parameter models. Among three-parameter models, retention description introduced by Horváth and Liang provided comparable quality of regression at significantly improved robustness. Multivariate analysis of the best three-parameter models provided the description of physicochemical properties of dopamine precursors and metabolites. Principal component analysis and logistic regression allowed structural characterization of dopamine-related compounds based solely on regression parameters extracted from an isocratic elution data. Both polarity and type of functional groups has been correctly assigned for 3-methoxytyramine that has not been part of an evaluation study. Among applied dual-retention models, Horváth´s model, initially developed to describe a retention of ionic compounds on nonpolar stationary phases, provided robust regression of experimental data and allowed an extraction of structural characteristics of dopamine-related compounds.

Are we approaching a post-monolithic era?

Thirty years after their introduction, monolithic stationary phases are an important member of chromatographic phases. When compared to conventional particulate materials, the continuous internal structure of both inorganic silica and organic polymer monoliths allows some hydrodynamic and analytical possibilities that are not provided by conventional particulate stationary phases. Polymer-based monolithic stationary phases offer simple preparation and straightforward surface modification, which makes them very versatile materials that are applicable, for example, as chromatographic stationary phases, sample enrichment units, enzymatic reactors, and external trigger-responding materials. On the other hand, current polymer monoliths cannot compete with efficiency provided by superficially porous and sub 2 µm particles. In this highlight article, I take advantage of the 30th anniversary of their introduction to discuss several concerns related to polymer-based monolithic stationary phases. Particularly, I focus on preparation repeatability, porous properties, swelling of the polymers in organic solvents, column efficiency for small molecules, and heterogeneity of dominant flow-through pores. In the end, I offer three possible approaches on how to overcome drawbacks related to stationary phases heterogeneity to further increase the applicability of polymer-based monolithic stationary phases.

Multichannel separation device with parallel electrochemical detection.

A device with four parallel channels was designed and manufactured by 3D printing in titanium. A simple experimental setup allowed splitting of the mobile phase in four parallel streams, such that a single sample could be analysed four times simultaneously. The four capillary channels were filled with a monolithic stationary phase, prepared using a zwitterionic functional monomer in combination with various dimethacrylate cross-linkers. The resulting stationary phases were applicable in both reversed-phase and hydrophilic-interaction retention mechanisms. The mobile-phase composition was optimized by means of a window diagram so as to obtain the highest possible resolution of dopamine precursors and metabolites on all columns. Miniaturized electrochemical detectors with carbon fibres as working electrodes and silver micro-wires as reference electrodes were integrated in the device at the end of each column. Experimental separations were successfully compared with those predicted by a three-parameter retention model. Finally, dopamine was determined in human urine to further confirm applicability of the developed device.

Intramedullary nailing of displaced four-part fractures of the proximal humerus.

BACKGROUND: Intramedullary nailing is already established technique for the treatment of two and three-part fractures of proximal humerus. The aim of the study was to prospectively evaluate the efficacy and functional outcome after treatment of four-part fractures of proximal humerus with Multiloc proximal humeral nail. DESIGN: prospective monocentric cohort study. SETTING: single level 1 traumacenter. MATERIALS AND METHODS: From February 2011 to March 2016, 40 patients with displaced four-part proximal humeral fractures were treated with intramedullary nail inserted through anterolateral approach. Minimum one year follow up completed 35 patients and were involved into the study. RESULTS: After mean follow up period of 25.8 months 29 of 35 fractures healed. Average absolute Constant score in all 35 patients reached 57.7 points, relative side related Constants score 66.8% of contralateral extremity. Together there were 20 complications. in 6 cases (17%) developed complete avascular necrosis of the head. One deep infection was treated by implantation of antibiotic cement discs. Twelve secondary surgeries were performed, mostly for avascular necrosis development. Function and pain were significantly influenced by the quality of fracture reduction (p < 0.05) and development of complete AVN (p = 0.001). Group of 29 patients without AVN reached relative Constant score 73% of contralateral extremity. CONCLUSIONS: Intramedullary nailing can be used as possible fixation technique for the treatment of four-part fractures of proximal humerus. In experienced hands provides nailing osteosynthesis similar results as reconstruction with locking plates. Appropriate reduction of fracture fragments is the key for good functional result. LEVEL OF EVIDENCE: Level 2b - monocentric prospective cohort study.

Can Occurrence of Pesticide Metabolites Detected in Crops Provide the Evidence on Illegal Practices in Organic Farming?

Modern pesticides rapidly degrade after their application due to both physicochemical factors and through biotransformation. Consequently, pesticide residues in samples might be either undetectable or detected at low concentrations (≤10 µg/kg). Under such conditions, a monitoring of pesticide metabolites in samples might be a conceivable solution enabling the documentation of earlier pesticide use. Analysis of metabolites might pose analytical challenges because pesticide degradation leads to the production of a number of metabolites, differing somewhat in their structure and polarity. This study was focused on the determination of pesticide residues and their metabolites in samples of grapevine and wine using ultrahigh-performance liquid chromatography coupled with high-resolution mass spectrometry, with the objective of supporting the possibility of the verification of the method of farming. It documents the identification of pesticide metabolites commonly used in conventional farming and provides a characterization of pesticide degradation during grapevine growth, maturation, and during the wine-making process.