Electrophoretic Techniques Research Articles

A Case for Co-oximetry: Methemoglobinemia secondary to a Rare Hemoglobin Variant

Abstract Introduction/Objective The presence of dyshemoglobins and some inherited hemoglobin (Hb) variants can cause over or underestimation of oxygen saturation (O2sat) when using a pulse oximeter (ox). These noninvasive medical devices rely on two wavelengths, a pulsing arterial bed, and light absorption properties of Hb to measure the difference between deoxy- and oxy-Hb. In contrast, a co-oximeter (co-ox) requires arterial blood. By using at least four wavelengths, co-ox measures the functional and fractional O2sat to detect all forms of Hb, including those that do not bind oxygen (O2), such as methemoglobin (metHb). Methemoglobinemia may be congenital or acquired, resulting from exposure to certain toxins, metabolic disorders, enzymatic deficiencies such as cytochrome b5 reductase deficiency, or hemoglobinopathies. Methods/Case Report A healthy, adolescent male undergoing a sports physical at his pediatrician’s office was hypoxic by pulse ox and sent to the emergency department (ED) for evaluation. On exam, he had cold, blue tinged fingertips and lips but was otherwise asymptomatic. His O2sat was 88-90% on room air with minimal improvement with 6L of supplemental O2. Co-ox revealed a metHb level > 20%. The father also mentioned that the mother had frequent ED visits for hypoxia. Capillary Hb electrophoresis showed peaks in the HbA, HbF, and HbA2 zones of 78.3%, 17.9%, and 3.6%, respectively. All CBC parameters were within the reference intervals. The large peak in the HbF zone would suggest hereditary persistence of fetal hemoglobin (HPFH); however, the clinical picture was not consistent. Further reference laboratory investigation determined that the patient was heterozygous for Hb Tübingen (HbTüb), a beta globin chain variant resulting from a point mutation at position 106. By mass spectrometry, the variant quantified as 38% of the total hemoglobin, indicating underestimation by electrophoretic techniques. Results (if a Case Study enter NA) NA Conclusion This case presents an essentially asymptomatic patient with hypoxia and elevated methemoglobin levels. Electrophoretic hemoglobinopathy and CBC workup were clinically inconsistent with HPFH and the presence of an aberrant hemoglobin migrating in the HbF must be considered in the differential diagnosis. The manifestation of HbTüb as methemoglobinemia in this context adds complexity to the diagnostic puzzle. Further laboratory exploration into the underlying mechanisms driving this unique presentation is crucial for a comprehensive understanding and tailored management approach.

Green synthesis of Oscimum Sanctum mediated silver nanoparticles to fabricate sensors for hydrogen peroxide detection

In this study, silver nanoparticles (Ag NPs) of an average size of ∼15 nm have been produced using the green synthesis technique with Oscimum Sanctum leaf extract. Further, these nanoparticles have been used to prepare electrodes for electrochemical sensors to monitor hydrogen peroxide. Hydrogen peroxide sensors have many applications in different sectors, such as medical, agriculture, and industry. The optical and structural characteristics of the Ag NPs have been investigated by UV–visible absorption spectroscopy and XRD patterns. Particle size, shape, and morphology of as-synthesized Ag NPs have been analyzed using transmission and scanning electron microscopic techniques. These nanoparticles were deposited onto an indium-tin-oxide glass substrate using the electrophoretic technique, which was used as an electrode to assess the electro-oxidation of Ag NPs by cyclic voltammetry. The sensitivity of the sensor has been estimated to be 4.16 µA/mM-cm2. The limit of detection of the sensor is estimated to be 40 µM within the linear range of 5–28 mM. The lattice parameter, interplanar spacing, and crystallite size of Ag NPs have been quantified and estimated against pH variation.

Monosaccharide-Mediated Glycoxidation of Bovine Serum Albumin and Its Prevention by Nigella sativa.

The substantial rise in metabolic illnesses that has occurred in both developed and developing countries over the last three decades has been linked to an increase in sugar-added foods and sweetened beverage intake. The significance of advanced glycation end products (AGEs) in the pathophysiology of metabolic diseases related to modern nutrition is an emerging issue. Spices and herbs can potentially be potent AGE production inhibitors due to their high polyphenol content. The inhibitory activity of an aqueous extract of Nigella sativa seeds (NS) on glucose- and fructose-mediated glycation of bovine serum albumin (BSA) was investigated. The glycation of proteins and its prevention using NS were assessed using spectrophotometry, spectrofluorometrics, and electrophoretic techniques. Additionally, the NBT assay, DNPH assay, Ellman assay, and thioflavin T assay were used to observe the biochemical alterations caused by glycated BSA. Molecular docking was employed to dock the BSA active site residues with inhibitors. Our data showed that NS protects against glucose- and fructose-mediated glycation and aggregation in vitro by inhibiting the formation of fructosamine, protein carbonyl content, free sulfhydryl groups, and fluorescent AGEs. Furthermore, NS also inhibited the production of β-cross-amyloid aggregates in proteins. It was interesting to note that the inhibition was found to be significantly higher in the Glu-BSA system, although the glycation product formed in the Fru-BSA system was higher compared to the Glu-induced protein system. It can be concluded that, by inhibiting AGE production, oxidation, and aggregation of the protein, NS may be an effective antiglycation drug for the prevention of diabetes complications.

Application of electrospray triple quadrupole mass spectrometry in assessing thalassemia in a population.

Thalassemia is widely prevalent in Asian countries and the Middle East region of the world. While the prevalence of α thal is around 5-20% in the population, the incidence of β thal is around 3-4%. The available data on thalassemia in Indians is based on chromatographic or electrophoretic techniques. The present study demonstrates the utility of mass spectrometry in detecting thalassemia in a small, healthy population from Tiptur, Karnataka. The study included a population of 315 individuals, spanning both children and adults, with ages ranging from 4 to 76 years. EDTA whole blood was collected after obtaining their written consent. The intensities of various globin chains were measured using a triple quadrupole mass spectrometer. The ratio of the peak areas of globin chains (α/β, δ/β (%)) served as biomarkers to detect thalassemia. Thirty random samples of α thalassemia were analyzed using GapPCR. The prevalence of a thal is 21.65% (68/314) based on α/β (< =0.8) and the prevalence of β thal is 3.8% (12/314) based on δ/β (%) (> =11.0). GapPCR detected three heteromutants of α deletion, one mutation in the HBA1 gene, and one a triplication (16.7%, 5/30). One sample was excluded from the analysis due to the presence of a variant. The utility of mass spectrometry for screening thalassemia in a population is demonstrated. The higher occurrence of α-thalassemia in the studied population underscores the importance of screening for thalassemia in regions with reportedly high prevalence rates.

Gluten Is Not Gluten.

Wheat gluten is responsible for the unique baking properties of wheat flour, but it also causes wheat-related disorders in predisposed individuals. Different commercially available gluten materials are commonly used for a variety of assays, but a detailed characterization of their composition is missing in many cases. This is why we aimed to provide an in-depth analysis of three commonly used gliadin and gluten materials from two different batches using gel electrophoretic and chromatographic techniques. The gliadin material did not show the typical qualitative and quantitative protein composition and does not appear to be representative of wheat gliadin. The two gluten materials had the expected protein composition, but both showed large batch-to-batch variability regarding total protein content. Since these variations result in different biochemical, immunological, and functional behaviors, it is important to analyze at least the total protein content of each material and each batch.

Impact of Ultrafiltration on the Physicochemical Properties of Bovine Lactoferrin: Insights into Molecular Mass, Surface Morphology, and Elemental Composition

The large-scale isolation of bovine lactoferrin (bLF) typically involves using large amounts of concentrated eluents, which might introduce impurities to the final product. Sometimes, protein pre-concentration is required for the greater accuracy of experimental results. In this research, the supplied bLF sample was subjected to additional ultrafiltration (UF) to eliminate possible small impurities, such as salts and peptides of bLF. Beforehand, the basic characterization of native bLF, including surface-charge properties and the structural sensitivity to the various pH conditions, was performed. The study aimed to evaluate the difference in molecular mass, primary structure, surface morphology, and elemental composition of the protein before and after UF. The research was provided by application of spectroscopic, spectrometric, electrophoretic, and microscopic techniques. The evident changes in the surface morphology of bLF were observed after UF, while the molecular masses of both proteins were comparable. According to MALDI-TOF/MS results, UF had a positive impact on the bLF sample representation, improving the identification parameters, such as sequence coverage and intensity coverage.

The High-Molecular-Weight Glutenin Subunits of the T. timopheevii (AuAuGG) Group.

Polyploid wheats include a group of tetraploids known as Timopheevii (AuAuGG), which are represented by two subspecies: Triticum timopheevii ssp. timopheevii (cultivated) and Triticum timopheevii ssp. araraticum (wild). The combined use of electrophoretic (SDS-PAGE) and chromatographic (RP-HPLC) techniques carried out on high-molecular-weight glutenin subunits (HMW-GSs) permitted the association of different x- and y-type subunits to the A and G genomes and the assessment of allelic variation present at corresponding loci. The results also revealed that in both subspecies, accessions are present that possess expressed y-type subunits at the Glu-A1 locus. Genes corresponding to these subunits were amplified and amplicons corresponding to x- and y-type genes associated with the A genome were detected in all accessions, including those without expressed x- and y-type subunits. The comparison with genes of polyploid wheats confirmed the structural characteristics of typical y-type genes, with the presence of seven cysteine residues and with hexapeptide and nonapeptide repeat motifs. The identification of wild and cultivated T. timopheevii with both x- and y-type glutenin subunits at the Glu-A1 and Glu-G1 loci represents a useful source for the modification of the allelic composition of HMW-GSs in cultivated wheats with the ultimate objective of improving technological properties.

Demonstration of Physicochemical and Functional Similarity of Biosimilar Pegfilgrastim-cbqv to Pegfilgrastim.

Pegfilgrastim-cbqv/CHS-1701 (UDENYCA®) (hereafter referred to as pegfilgrastim-cbqv) was approved in 2018 by the US Food and Drug Administration as a biosimilar for pegfilgrastim (Neulasta®) (hereafter referred to as pegfilgrastim). Both pegfilgrastim-cbqv and pegfilgrastim are conjugates of recombinant human granulocyte colony stimulating factor (r-metHuG-CSF) with a 20kDa polyethylene glycol (PEG) indicated to decrease the incidence of infection, as manifested by febrile neutropenia, in patients receiving myelosuppressive anticancer drugs. The demonstration of analytical similarity for PEG-protein conjugates presents unique challenges since both the protein and PEG attributes must be characterized. The current study demonstrates the analytical similarity of pegfilgrastim-cbqv and the reference product, pegfilgrastim. In addition to the physicochemical and functional characterization of the protein, the study assessed attributes specific to PEGylation including PEG size and polydispersity, site of attachment, linker composition, and PEGylation process-related variants. The structural, functional, and stability attributes of pegfilgrastim-cbqv and pegfilgrastim were compared using state-of-the-art analytical methods. For the protein, the primary structure, disulfide structure, and secondary and tertiary structures were assessed using traditional protein characterization techniques such as mass spectrometry (MS), circular dichroism (CD), intrinsic fluorescence, and differential scanning calorimetry (DSC), as well as more advanced techniques such as two-dimensional (2D) nuclear magnetic resonance (NMR) and hydrogen deuterium exchange (HDX). For the PEG moiety, the site of attachment, occupancy, linker composition, size and polydispersity were compared using mass spectrometry (both intact and after endoprotease digestion), multiangle light scattering detection (MALS), and Edman degradation. Purity assessments included the assessment of both protein variants and PEGylation variants using chromatographic and electrophoretic analytical separation techniques. The functional similarity between pegfilgrastim-cbqv and pegfilgrastim was compared using both a cell-based bioassay and surface plasmon resonance (SPR). The degradation rates and stability profiles were compared under accelerated and stressed conditions. Biosimilarity was demonstrated by a thorough assessment of physiochemical and functional attributes, as well as comparative stability, of pegfilgrastim-cbqv relative to pegfilgrastim. These studies demonstrated identical primary structure and disulfide structure, highly similar secondary and tertiary structure, as well as functional similarity. The impurity profile of pegfilgrastim-cbqv was comparable to that of pegfilgrastim with only minor differences in PEGylation variants and a slight offset in the PEG molar mass. These differences were not clinically relevant. The degradation profiles were qualitatively and quantitatively similar under accelerated and stress conditions. The structural, functional, and stability data demonstrate that pegfilgrastim-cbqv is highly similar to the reference product, pegfilgrastim.

Environmental Pollution Monitoring via Capillary Zone Electrophoresis and UHPLC Simultaneous Quantification of Some Antipsychotic Drug Residues in Industrial Wastewater Effluents

Monitoring and measuring pharmaceutical pollutants in environmental samples is a vital and complex task due to their potential detrimental effects on human health, even at low levels. Using capillary zone electrophoresis (CZE) and ultra-high-performance liquid chromatography (UHPLC), it was possible to separate and measure three commonly used antipsychotic drugs, chlorpromazine (CPZ), haloperidol (HAL), and risperidone (RIS), in wastewater of the pharmaceutical industry. The technique of solid-phase extraction (SPE) was developed and implemented as a very effective method for preparing samples prior to analysis. The settings of the capillary electrophoretic and chromatographic techniques were adjusted to obtain the most efficient separation profile for the medications being studied. The concentration of all the medicines being investigated ranged from 0.5 to 50 µg/mL. SPE was used to treat real wastewater samples after a thorough validation process that followed the rules set by ICH-Q2B. The developed assays were then effectively employed to identify the tested antipsychotic substances in the real wastewater samples. The provided methodologies may be efficiently utilized to monitor the extent of environmental contamination caused by the investigated pharmaceuticals.

Study the Structure and Properties of Antibacterial TiO2/HAP Coating onto Ti Alloy Using Electrophoretic Deposition Technique for Biomedical Application

In the present study, single layer, titanium dioxide (TiO2) and TiO2/HAP nano-particles coatings were fabricated by electrophoretic deposition on titanium alloy Ti-6Al-4V. As a result of accidents, diseases and other causes, efforts and research are continued to reach results closest. The damaged that occur to some of them in the living of the organism in general and the human being in particular, this can be done throughout the existing the implantation of medical minerals, ligaments of bones, teeth and other parts that compensate. Through science, opportunities and experiments have been provided to obtain satisfactory results in terms of the biocompatibility of titanium alloy Ti-6Al-4V implants and their alloys, which are classified as one of the best biocompatible materials within vivo tissues. This study aims to increase improve this alloy antibacterial properties by coating the surface with a bio ceramic (TiO2 and TiO2/HAP) nano-powders. were made on titanium alloy Ti-6Al-4V substrate using an electrophoretic deposition (EPD) technique, charged particles suspended in ethanol at a concentration of 50 g/L, for each powder with ideal conditions of 20V and a deposition time of (4 min). The surface characteristics of coated substrates, such as micro-hardness, surface roughness and wettability antibacterial test were evaluated and compared to that of the uncoated substrates. The results showed that the EPD is a promising technique to create a bio coating on titanium alloy Ti-6Al-4V substrate with outstanding structure and properties for biomedical applications. The results demonstrated that the coated sample under coating conditions (20V, 4min) which is close to the ratio found in living bone. The results also of the wettability test showed that the contact angle of the deposited paint is 5.9 degrees, and this positive result is useful for biomedical applications and proved that the coating is hydrophilic.

Surface Evaluation of Tricalcium Phosphate Bioceramic Coating on SS-316L by Electrophoretic Deposition

The development of orthopedic implant materials has become an important topic of discussion lately. The SS-316L alloy is widely used as an implant material due to its relatively low cost, corrosion resistance, and ease of production. However, metal alloys, especially SS-316L, are prone to ion release into the blood over time. Therefore, TCP or tricalcium phosphate [Ca3(PO4)2] is needed to coat the surface of SS-316L, preventing ion release into the blood and enhancing the biocompatibility of the implant material. In this study, TCP coating was applied to the SS-316L substrate using the electrophoretic deposition technique. The influence of deposition time on changes in microstructure and mechanical properties is the main focus of this study. The results of the coating technique indicate that the deposition yield increases with the deposition time. Morphological testing results show that increasing deposition time improves coating quality by increasing the thickness of the coating layer and preventing layer peeling. The coating process also reveals the accumulation of layers in certain areas and the formation of thin layers in other regions. A deposition time of 30 minutes results in a coating thickness ranging from 48.7 to 57.9 µm. Hardness testing, conducted with indentation loads of 50, 100, and 300 gf, indicates that longer deposition times and higher indentation loads during hardness testing result in reduced material hardness.

Effect of Air Classification and Enzymatic and Microbial Bioprocessing on Defatted Durum Wheat Germ: Characterization and Use as Bread Ingredient.

Its high dietary fiber and protein contents and nutritional quality make defatted wheat germ (DWG) a valuable cereal by-product, yet its negative impact on food structure limits its use as a food ingredient. In this research, DWG underwent air classification, which identified two fractions with high fiber (HF) and low fiber/high protein (LF) contents, and a bioprocessing protocol, involving treatment with xylanase and fermentation with selected lactic acid bacterial strains. The degree of proteolysis was evaluated through electrophoretic and chromatographic techniques, revealing differences among fractions and bioprocessing options. Fermentation led to a significant increase in free amino acids (up to 6 g/kg), further enhanced by the combination with xylanase. When HF was used as an ingredient in bread making, the fiber content of the resulting bread exceeded 3.6 g/100 g, thus reaching the threshold required to make a "source of fiber" claim according to Regulation EC No.1924/2006. Meanwhile, all breads could be labeled a "source of protein" since up to 13% of the energy was provided by proteins. Overall, bioprocessed ingredients lowered the glycemic index (84 vs. 89) and increased protein digestibility (80 vs. 63%) compared to control breads. Technological and sensory analysis showed that the enzymatic treatment combined with fermentation also conferred a darker and more pleasant color to the bread crust, as well as better crumb porosity and elasticity.

Serum mass spectrometry for treatment monitoring in patients with multiple myeloma receiving ARI0002h CAR T-cells.

Chimeric antigen receptor (CAR) T-cell therapies have increased the patients with relapsed/refractory multiple myeloma (RRMM) in whom standard electrophoretic techniques fail to detect the M-protein. Quantitative immunoprecipitation mass spectrometry (QIP-MS) can accurately measure serum M-protein with high sensitivity, and identify interferences caused by therapeutic monoclonal antibodies. Here, we investigate the outcome of QIP-MS in 33 patients treated with the academic BCMA-directed CAR T-cell ARI0002h (Cesnicabtagene Autoleucel). QIP-MS offered more detailed insights than serum immunofixation (sIFE), identifying glycosylated M-proteins and minor additional peaks. Moreover, the potential interferences owing to daratumumab or tocilizumab treatments were successfully detected. When analysing different assay platforms during patient's monitoring after ARI0002h administration, we observed that QIP-MS showed a high global concordance (78.8%) with sIFE, whereas it was only moderate (55.6%) with bone marrow (BM)-based next-generation flow cytometry (NGF). Furthermore, QIP-MS consistently demonstrated the lowest negativity rate across the different timepoints (27.3% vs. 60.0% in months 1 and 12, respectively). Patients with QIP-MS(+)/BM-based NGF(-) showed a non-significant shorter median progression free survival than those with QIP-MS(-)/BM-based NGF(-). In summary, we show the first experience to our knowledge demonstrating that QIP-MS could be particularly useful as a non-invasive technique when evaluating response after CAR T-cell treatment in MM.

Quality evaluation of highly purified human menopausal gonadotropin preparations by means of gel electrophoresis and mass spectrometry.

Human gonadotropins are glycoprotein hormones with a highly complex structure, which demands the application of sophisticated analytical methodologies to assess their quality. The principal objective of this study was a comparative evaluation of gel electrophoretic techniques and mass spectrometry-based methods for the quality study of the two urinary-derived, highly purified, human menopausal gonadotropin preparations, Menopur 75/75 I. U. and Meriofert 75 I. U. Molecular mass (Mr), isoelectric point (pI), and isoform pattern of studied compounds were estimated via SDS-PAGE and 2D gel electrophoresis, whereas matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used for the downstream characterization of peptides obtained after in-gel tryptic digestion of selected protein spots. Additionally, for the estimation of the glycosylation pattern of these biologics, the enzymatic release of oligosaccharides was performed, and the isoform pattern was studied. Gel electrophoresis showed a typical electrophoretic behaviour for protein biotherapeutics medicines consisting of extremely complex spot patterns migrating at different masses and pIs. MS analysis proved to be a powerful tool for the identification and detailed characterization of the gonadotropins and the relevant peptides were identified with high sequence coverages. The results of this study are not only useful for the quality assessment of this class of complex biopharmaceuticals but may also serve as a supporting platform for further development of biopharmaceuticals based on modulation of the glycosylation pattern to enhance efficacy or reduce side effects.

CONCENTRATION OF NANO FLUID/BASE FLUID SUSPENSION ENHANCE SURFACE CHARGE WITH PH STABILITY FOR LOW TO MEDIUM TEMPERATURE PHASE CHANGE MATERIALS

Zeta potential and poly-dispersivity are used to characterize the samples that is obtained using absorption refrigeration system for low to medium temperature phase transition materials. Salicyclic acid, Benzanilide, Hydroquinone, Potassium thiocyanates, D-mannitol, Alunimium oxide, Iron oxide, and ZnO active concentration with base fluid, aspects including the influence of the PCMs property based on their phase transition mutual interaction are explored. In order to comprehend their behavior and improve their performance, functional materials synthesis and characterization depend heavily on the isoelectric point. Understanding the material surface charge role of the medium's pH stability to the many liquid-phase procedures involved in the synthesis of materials, since it conduct the processes like agglomeration, coagulation, peptization to form solid particles materials. Zeta potential measure, which commonly use concentration of volume fraction methods, electrophoretic migration techniques, are hence a valuable source of data.

Metal Complexes in Biology and Medicine the System Cadmium (II) / Iron (II) /Zinc (II) - Proline

Metal ions are fundamental elements for the maintenance of the life span’ s of the humans, animals and plants. In coordination compound studies, a knowledge of the magnitude of the stability constants of complexes is necessary for preliminary quantitative treatment. Present work described method that involves the use of paper electrophoretic technique for the study of the equilibria in binary complex systems in solution. This method is based on the movement of a spot of metal ion in an electric field at various pHs of the background electrolyte. A plot of overall mobility of metal / complex ion versus pH was used to obtain information on the formation of binary complexes and to calculate their stability constants. The stability constant of the ML and ML2 complexes of cadmium (II) – proline,iron(II) – proline and zinc(II) – proline, have been found to be (4.61 ± 0.01; 3.04 ± 0.03), (4.21 ± 0.01; 2.89 ± 0.09) and (4.98 ± 0.02; 2.44 ± 0.05) (logarithm constant values), respectively at 35 ºC and ionic strength 0.1 mol L-1. The first and second stability constants follow the order zinc (II) &gt;cadmium (II) &gt; iron (II). Metal complexes can offer their action such as anti-inflammatory, antidiabetic, antimicrobial, anticancer and ant thyroid compounds. Metal based drugs bioactivity can be increase by metal chelation, which in turn increase their absorbance and stability.

Implication of metal complexes in biology and medicine, the system cadmium (II)/iron (II)/zinc (II) – sarcosine

Implication of metal complexes in biology and medicin, the system cadmium (II)/iron (II) / zinc (II) – sarcosine. A technique involving the use of paper Electrophoretic Technique is described for the study of equilibria in binary complex system in solution. This methodis based upon the migration of a spot of metal ion on a paper strip at different pH values of background electrolyte containing 0.1 mol L -1 perchloric acid and 0.01 mol L -1 sarcosine. A graph of pH against mobility gives information about the binary complexes and permit to calculate their stability constants. The first and second stability constants of [Cd (II) – sarcosine], [Fe (II) – sarcosine] and [Zn (II) – sarcosine] complexes were found to be (3.94 ± 0.03; 2.66 ± 0.07), (3.70 ± 0.01; 2.54 ± 0.03) and (4.39 ± 0.01; 2.74 ± 0.07) logarithm stability constant values for Cd (II), Fe (II) and Zn (II) complexes, respectively at ionic strength 0.1 Mol L -1 and temperature of 35 0 C. The stability constants follow the order: Cd (II) &gt; Fe (II) &gt; Zn (II).

Electrophoretic Deposition of 58S Bioactive Glass- Polymer Composite Coatings on 316L Stainless Steel: An Optimization for Corrosion, Bioactivity, and Cytocompatibility.

This study presents a facile fabrication of 58S bioactive glass (BG)-polymer composite coatings on a 316L stainless steel (SS) substrate using the electrophoretic deposition technique. The suspension characteristics and deposition kinetics of BG, along with three different polymers, namely ethylcellulose (EC), poly(acrylic acid) (PAA), and polyvinylpyrrolidone (PVP), have been utilized to fabricate the coatings. Among all coatings, 58S BG and EC polymers are selected as the final composite coating (EC6) owing to their homogeneity and good adhesion. EC6 coating exhibits a thickness of ∼18 μm and an average roughness of ∼2.5 μm. Herein, EC6 demonstrates better hydroxyapatite formation compared to PAA and PVP coatings in simulated body fluid-based mineralization studies for a period of 28 days. Corrosion studies of EC6 in phosphate-buffered saline further confirm the higher corrosion resistance properties after 14 days. In vitro cytocompatibility studies using human placental mesenchymal stem cells demonstrate an increase in cellular viability, attachment, and higher proliferation compared to the bare SS substrate. EC6 coatings promote osteogenic differentiation, which is confirmed via the upregulation of the OPN and OCN genes. Moreover, the EC6 sample exhibits improved antibacterial properties against Escherichia coli and Staphylococcus aureus compared to the uncoated ones. The findings of this work emphasize the potential of electrophoretically fabricated BG-EC composite coatings on SS substrates for orthopedic applications.

Performance Characteristics and Limitations of the Available Assays for the Detection and Quantitation of Monoclonal Free Light Chains and New Emerging Methodologies.

Light chain measurements form an essential component of the testing strategy for the detection and monitoring of patients with suspected and/or proven plasma cell disorders. Urine-based electrophoretic assays remain at the centre of the international guidelines for response assessment but the supplementary role of serum-free light chain (FLC) assays in response assessment and the detection of disease progression due to their increased sensitivity has been increasingly recognised since their introduction in 2001. Serum FLC assays have also been shown to be prognostic across the spectrum of plasma cell disorders and are now incorporated into risk stratification scores for patients with monoclonal gammopathy of undetermined significance (MGUS), smouldering multiple myeloma, and light chain amyloidosis (AL amyloidosis), as well as being incorporated into the criteria for defining symptomatic multiple myeloma. There are now multiple different commercially available serum FLC assays available with differing performance characteristics, which are discussed in this review, along with the implications of these for patient monitoring. Finally, newer methodologies for the identification and characterisation of monoclonal FLC, including modifications to electrophoretic techniques, mass spectrometry-based assays and Amylite, are also described along with the relevant published data available regarding the performance of each assay.

Directing in Vitro Selection towards G-quadruplex-forming Aptamers to Inhibit HMGB1 Pathological Activity.

In the search for novel, effective inhibitors of High-Mobility Group Box1 (HMGB1)-a protein involved in various inflammatory and autoimmune diseases as well as in cancer-we herein discovered a set of anti-HMGB1 G-quadruplex(G4)-forming aptamers by using an in vitro selection procedure applied to a doped library of guanine-rich oligonucleotides. The selected DNA sequences were then studied in a pseudo-physiological buffer mimicking the extracellular medium, where HMGB1 exerts its pathological activity, using spectroscopic, electrophoretic, and chromatographic techniques. All the oligonucleotides proved to fold into monomeric G4s and in some cases also dimeric species, stable at physiological temperature. Remarkably, the protein preferentially recognized the sequences forming dimeric parallel G4 structures, as evidenced by a properly designed chemiluminescent binding assay which also highlighted a good selectivity of these aptamers for HMGB1. Moreover, all aptamers showed anti-HMGB1 activity, inhibiting protein-induced cell migration. The acquired data allowed identifying L12 as the best anti-HMGB1 aptamer, featured by high thermal and enzymatic stability, no toxicity at least up to 5 μM concentration on healthy cells, along with potent anti-HMGB1 activity (IC50 ca. 28 nM) and good binding affinity for the protein, thus indicating it as a very promising lead candidate for in vivo studies.