Recent biopharmaceutical applications of capillary electrophoresis methods on recombinant DNA technology-based products.

Biopharmaceuticals (recombinant technology-based products, vaccines, whole blood and blood components, gene therapy, cells, tissues, etc.,) are described as biological medical products produced from various living sources such as human, microbial, animal, and so on by manufacturing, extraction, or semi-synthesis. They are complex molecules having high molecular weights. For their safety and efficacy, their structural, clinical, physicochemical, and chemical features must be carefully controlled, and they must be well characterized by analytical techniques before the approval of the final product. Capillary electrophoresis (CE) having versatile modes can provide valuable safety and efficacy information, such as amino acid sequence, size variants (low and high molecular weight variants), charged variants (acidic and basic impurities), aggregates, N-linked glycosylation, and O-linked glycosylation. There are numerous applications of CE in the literature. In this review, the most significant and recent studies on the analysis of recombinant DNA technology-based products using different CE modes in the last ten years have been overviewed. It was seen that the researches mostly focus on the analysis of mAbs and IgG. In addition, in recent years, researchers have started to prefer CE combined mass spectrometry (MS) techniques to provide a more detailed characterization for protein and peptide fragments.

Fluorescence chemosensing of meldonium using a cross-reactive sensor array.

In this paper, we report a fluorescent sensor array approach for the urinary detection of a prohibited substance in sports, meldonium. Four chemosensors with ethidium bromide scaffolds were employed in this method. The interaction between meldonium and chemosensors was investigated by different techniques, such as ultraviolet-visible absorption and fluorescence spectroscopy, nuclear magnetic resonance, and mass spectrometry. Molecular dynamics simulation was also used to elucidate and support the interaction mechanisms between meldonium and the chemosensors. Differential responses obtained from the sensor array enabled the qualitative and quantitative analyses of meldonium with low error values. This method was able to detect and quantify meldonium at the nM level, fulfilling the requirements of minimum performance defined by the World Anti-Doping Agency.

A New Comorbidity in Female Patients With Ankylosing Spondylitis: Pelvic Organ Prolapse.

AIM: The aim of this study was to investigate whether increased intra-abdominal pressure caused by pelvic inflammation and frequent use of the Valsalva maneuver increases the incidence of pelvic organ prolapse (POP) among female patients with ankylosing spondylitis (AS). METHODS: Thirty-nine patients diagnosed as having AS through the use of the modified New York criteria, 47 patients with chronic low-back pain (CLBP), and 38 healthy controls (HCs) were included in this study. All the patients and the HCs underwent thorough physical and gynecological examinations. Pelvic organ prolapse was graded blindly by a gynecologist. Presence or absence of cystocele (CS), rectocele (RC), and uterine prolapse (UP) was noted. RESULTS: The incidences of CS, RC, and UD were significantly higher among the AS patients compared with the HCs (p = 0.001, 0.026, and 0002, respectively). The incidences of CS (p = 0.042) and UD (p = 0.017) were significantly higher among the AS patients compared with the CLBP patients. CONCLUSIONS: The incidence of POP is higher among patients with AS compared with normal population. These patients should be questioned about the symptoms of POP and recommended, if necessary, regular gynecological examinations, as well as specific exercises including those targeting AS.

Simultaneous determination and validation of emtricitabine, rilpivirine and tenofovir from biological samples using LC and CE methods.

A combination of antiretroviral agents is frequently used in effective treatment of the human immunodeficiency virus infection. In this study, two different separation methods are presented for the simultaneous determination of emtricitabine, rilpivirine and tenofovir from raw materials and urine samples. Developed liquid chromatography and capillary electrophoresis methods were thoroughly optimized for high analytical performances. Optimization of multiple variables at the same time by performing a minimum number of experiments was achieved by the Box-Behnken design, which is an experimental design in response surface methodology, in capillary electrophoresis. The results of the experimental design ensure minimum analysis time with well-separated analytes. Separation conditions, such as different stationary phases, pH level, organic modifiers and temperatures in liquid chromatography method, were also optimized. In particular, among stationary phases, the core-shell column especially enhanced the effectiveness of separation in liquid chromatography. Both methods were fully validated and applied to real samples. The main advantage of the developed methods is the separation of the drug combination in a short time with high efficiency and without any time-consuming steps.

Detection and quantification of ATP in human blood serum.

Two fluorometric sensors based on the tri-serine tri-lactone scaffold and thiourea or sulfonamide moieties serving as hydrogen bond donors allowing for anion binding are described. The sensor utilizing thiourea as a recognition moiety shows fluorescence enhancement while the sensor with sulfonamide shows quenching upon addition of phosphates. Sensor arrays composed of two sensors are able to discriminate structurally similar organic phosphates in the presence of interferents in human blood serum. The quantitative analysis of ATP in human blood serum shows high accuracy (the root mean square error of prediction, 1.65%) without requiring any sample pretreatment.

Surface-enhanced Raman probe for rapid nanoextraction and detection of erythropoietin in urine.

We present a surface-enhanced Raman probe (SERS) platform for the determination of a prohibited substance, recombinant erythropoietin (rEPO), in urine matrix, using nanoparticles as substrate. Rod-shaped gold nanoparticles (GNR) were modified with a Raman label and an antibody as SERS probe. We developed two SERS-based immunoassays for detection and quantification of rEPO in urine. In the first assay, rEPO was determined by a sandwich assay with gold surfaces and GNR. In the second assay, rEPO was extracted by using core shell-structured magnetic iron oxide gold nanoparticles, and again sandwich assay was performed by using GNR. We also demonstrated the ability of the proposed method to discriminate rEPO and urinary erythropoietin (uEPO). A good linear correlation was obtained between logarithms of rEPO concentrations in urine and Raman intensities within the range of 10-1-103 pg mL-1 rEPO concentrations. Detection limits which are smaller than 0.1 pg mL-1 levels were achieved owing to the high extractive performance of the nanoextraction techniques. Graphical Abstract Schematic represantation of surface-enhanced Raman probe for rapid nanoextraction and detection of erythropoietin.

Elevated serum levels of calprotectin (MRP8/MRP14) in patients with Behçet's disease and its association with disease activity and quality of life.

BACKGROUND: Behçet's disease (BD) is an inflammatory disease with multisystem chronic vasculitis. The disease is characterized by attacks of oral and genital ulcerations, skin lesions, arthritis, uveitis and deep vein thrombosis. The main histopathological feature is known to be vascular inflammatory change. Calprotectin (MRP8/MRP14) has been identified as an important alarmin that is expressed by activated phagocytes, granulocytes, monocytes and vascular endothelial cells, recognized by toll-like receptors, and induces a thrombogenic and inflammatory response in human microvascular endothelial cells. AIM: We aimed to investigate the serum levels of calprotectin in patients with BD and its association with disease activity and quality of life. MATERIALS AND METHODS: Forty-eight patients (25 males and 23 females) and 47 healthy controls (29 males and 18 females) were included to study. BD Current Activity Form (BDCAF) was used to assess the disease activity of patients with BD. Quality of life was assessed by using the Nottingham Health Profile (NHP). Depression and anxiety symptoms were assessed by using the Hospital Anxiety and Depression Scale (HADS). Serum level of calprotectin was determined using an ELISA kit. Results. Serum levels of calprotectin was significantly higher in patients with BD compared to healthy controls (p = 0.001). Serum levels of calprotectin did not correlate with the sores of BDCAF, NHP and HADS. CONCLUSION: Calprotectin may play a significant role in the pathogenetic mechanisms of BD. Further insight into this area of research might provide opportunities to develop novel treatment strategies.

Colchicine levels in chronic kidney diseases and kidney transplant recipients using tacrolimus.

BACKGROUND: Tacrolimus is a CYP3A4 inhibitor and can alter colchicine metabolism. In this study, we aimed to evaluate plasma colchicine levels in different stages of kidney disease as well as in kidney transplant (KTx) recipients using tacrolimus. METHOD: This study included six familial Mediterranean fever (FMF) patients with normal glomerular filtration rate (GFR) as controls, three patients with low GFR, six FMF patients on hemodialysis (HD), and six FMF patients who were KTx recipients using tacrolimus. After a three-d washout period, plasma colchicine levels were measured at 0 (pre-dose), one, two, four, eight, and 24 h post-dose of 1 mg oral colchicine. Area under the curve 0-24 h (AUC0-24 ) and maximum concentration (Cmax ) were evaluated and compared between the groups. RESULTS: Colchicine AUC0-24 was six-fold higher in HD (p < 0.001) and three-fold higher in KTx recipients (p < 0.001) when compared to the control. The low GFR group had mildly higher AUC0-24 than the control group. Cmax levels were also higher in HD (p = 0.011) and KTx recipient (p = 0.06) groups and mildly elevated in low GFR patients in comparison with controls. CONCLUSION: Colchicine AUC0-24 and Cmax were significantly increased in HD patients and KTx recipients using tacrolimus. Therefore, dose adjustments are needed to avoid toxicity in both circumstances.

Carvacrol prevents methotrexate-induced renal oxidative injury and renal damage in rats.

PURPOSE: The purpose of this study was to investigate the effect of carvacrol (CAR) on methotrexate (MTX)-induced renal damage in rats. METHODS: Twenty-four male rats were equally divided into three groups: group I, control treatment; group II, MTX-treated; and group III, MTX+CAR-treated. A single dose of CAR (73 mg/kg) was administered intraperitoneally to group III on the first day of the experiment and a single dose of MTX (20 mg/kg) was administered intraperitoneally to groups II and III on the second day of the experiment. Blood samples and kidney tissue were obtained from each animal on day 8 for the measurement of malondialdehyde (MDA), total oxidant status (TOS), total antioxidant status (TAS), and oxidative stress index (OSI). Light microscopy was used for histopathological examination of kidney specimens. RESULTS: MDA, TOS and OSI levels were significantly greater in the group receiving MTX alone relative to the control animals, while the TAS level was significantly reduced in the MTX group compared with the control group. The administration of CAR was associated with significantly decreased MDA, TOS, and OSI levels and increased TAS levels relative to the rats treated with MTX alone. Animals treated with CAR exhibited decreased tubular degeneration and architectural impairment relative to animals treated with MTX alone; however, the difference in histological scores did not meet the threshold of statistical significance. CONCLUSIONS: MTX treatment results in oxidative damage to the rat kidney; damage which is partially abrogated by the administration of CAR.

Serum prolidase activity in benign joint hypermobility syndrome.

BACKGROUND: Moderate joint laxity is widespread in many joints of the body, and this condition is considered to be caused by an abnormality in the collagen structure. This study was carried out to determine the serum prolidase activity in female patients with benign joint hypermobility syndrome (BJHS), and to evaluate its correlation with their clinical features. METHODS: A total of 45 patients with BJHS and 40 healthy controls were included in the study. All of the patients with BJHS met the Beighton diagnostic criteria. All the patients and the control group underwent a comprehensive examination of the locomotor system and took the New York Posture Rating Test. The examination and test results were recorded. Serum prolidase activity was measured in both the groups. RESULTS: Prolidase activity was significantly lower in patients with BJHS (479.52 ± 126.50) compared to the healthy controls (555.97 ± 128.77) (p = 0.007). We found no correlation between serum prolidase activity and Beighton scores or New York rating test scores. On the other hand, mean prolidase activity was significantly lower in patients with pes planus or hyperlordosis compared to those without (p = 0.05, p = 0.03, respectively). We did not find such a correlation with the other clinical features. CONCLUSIONS: Significantly lower prolidase activity in patients with BJHS suggests that prolidase may affect the collagen metabolism and cause hyperlaxity.

Multivariate optimization of separation conditions for simultaneous determination of acemetacin and chlorzoxazone in a pharmaceutical preparation by HPLC using response surface methodology.

A new, fast, accurate, precise, and sensitive RP-HPLC method for the simultaneous determination of acemetacin and chlorzoxazone has been developed. Response surface methodology with a central composite design was used to optimize the acetonitrile and ammonium acetate percentage in the mobile phase and pH of ammonium acetate. The optimum separation was achieved on a C18 column (250 x 4.6 mm id, 5 microm particle size) using the mobile phase methanol-acetonitrile-0.02 M ammonium acetate, pH 9.4 (25 + 35 + 40, v/v/v) at a flow rate of 1.5 mL/min; UV detection at 270 nm, and cyanocobalamin as an internal standard. This developed method was validated and successfully applied to a coated tablet pharmaceutical preparation.

Boron-Doped Carbon Nanodots as a Theranostic Agent for Colon Cancer Stem Cells.

Carbon nanodots have drawn a great deal of attention due to their green and expedient opportunities in biological and chemical sciences. Their high fluorescence capabilities and low toxicity for living cells and tissues make them excellent imaging agents. In addition, they have a fluorimetric response against inorganic and organic species. Boron-doped carbon nanodots (B-CDs) with high fluorescence yield were produced from phenylboronic acid and glutamine as boron and carbon sources, respectively, by a hydrothermal method. First, the effects of the temperature on their fluorescence yield and the structural characteristics of B-CDs were investigated. Second, their cytotoxicity and cell death and proliferation behaviors were examined. The cytotoxicity was evaluated by the MTT assay. The cellular properties were evaluated with the distribution of caspase 3, Ki67, lamin B1, P16, and cytochrome c after the indirect immunoperoxidase technique. After the MTT assay, 1:1 dilution of all applicants for 24 h was used in the study. After immunohistochemical analyses, the application of B-CDs synthesized at 230 °C did not change control cell (Vero) proliferation, and also apoptosis was not triggered. Colo 320 CD133+ and CD133- cell-triggered apoptosis and cellular senescence were found to be synthesis temperature dependent. In addition, Colo 320 CD133- cells were affected relatively more than CD133+ cells from B-CDs. While B-CDs did not affect the control cells, the colon cancer stem cells (Colo 320 CD133+) were affected in a time-dependent manner. Therefore, the use of the synthesized B-CD product may be an alternative method for controlling or eliminating cancer stem cells in the tumor tissue.

SnO2 nanoparticles/waste masks carbon hybrid materials for DNA biosensor application on voltammetric detection of anti-cancer drug pazopanib.

This present study is the first investigation of pazopanib-dsDNA binding using bare and modified GCE. The interaction was mainly evaluated based on the decrease of voltammetric signal of deoxyadenosine by differential pulse voltammetry using three different ways, including the incubated solutions, dsDNA biosensor, and nanobiosensor. The nanobiosensor was fabricated with the help of SnO2 nanoparticles and carbon hybrid material. The carbon material is derived from the waste mask, the most used personal protective equipment for the ongoing COVID-19 pandemic. Both materials were synthesized via the green synthesis technique and characterized by various techniques, including BET, TEM, SEM-EDX, AFM, XPS, and XRD. Spectrophotometric and molecular docking studies also evaluated the pazopanib-dsDNA binding. All calculations showed that pazopanib (PZB) was active in the minor grove region of DNA.

Human hair rich in pyridinic nitrogen-base DNA biosensor for direct electrochemical monitoring of palbociclib-DNA interaction.

Carbon material derived from the waste-based biomass human hair (H), which is naturally rich in pyridinic nitrogen, provides a significant benefit in biosensor applications with its dominant conductivity character. The carbon material was synthesized from human hair waste by the hydrothermal carbonization (HTC) method, which is a promising green synthesis. A morphological characterization of the carbon materials was performed. In this study, H and amine-functionalized multi-walled carbon nanotubes (NH2-MWCNT) were combined for the first time as a modifier, which enhanced the glassy carbon electrode (GCE) surface area for deoxyribonucleic acid (DNA) biosensor studies. Palbociclib (PLB) is clinically used in the treatment of breast cancer. The novel electrochemical nanobiosensor was used to investigate the dsDNA-PLB interaction to evaluate the possibility that PLB causes conformational changes in DNA structure and/or oxidative damage. The interaction was conducted based on the voltammetric signals of deoxyguanosine (dGuo) and deoxyadenosine (dAdo) by differential pulse voltammetry (DPV) on a bare and H + NH2-MWCNT modified GCE. The proposed analytical method was applied to a pharmaceutical dosage form with a satisfactory recovery of 98.25 %. The nanobiosensor was tested in the presence of some interfering agents. The binding mechanism of dsDNA-PLB was also evaluated by spectroscopic and theoretical calculations.

Liquid chromatographic and spectrophotometric determination of diflucortolone valerate and isoconazole nitrate in creams.

A new RP-LC method and two new spectrophotometric methods, principal component regression (PCR) and first derivative spectrophotometry, are proposed for simultaneous determination of diflucortolone valerate (DIF) and isoconazole nitrate (ISO) in cream formulations. An isocratic system consisting of an ACE C18 column and a mobile phase composed of methanol-water (95 + 5, v/v) was used for the optimal chromatographic separation. In PCR, the concentration data matrix was prepared by using synthetic mixtures containing these drugs in methanol-water (3 + 1, v/v). The absorbance data matrix corresponding to the concentration data matrix was obtained by measuring the absorbances at 29 wavelengths in the range of 242-298 nm for DIF and ISO in the zero-order spectra of their combinations. In first derivative spectrophotometry, dA/dlambda values were measured at 247.8 nm for DIF and at 240.2 nm for ISO in first derivative spectra of the solution of DIF and ISO in methanol-water (3 + 1, v/v). The linear ranges were 4.00-48.0 microg/mL for DIF and 50.0-400 microg/mL for ISO in the LC method, and 2.40-40.0 microg/mL for DIF and 60.0-260 microg/mL for ISO in the PCR and first derivative spectrophotometric methods. These methods were validated by analyzing synthetic mixtures. These three methods were successfully applied to two pharmaceutical cream preparations.

Replacement of antibodies with bacteriophages in lateral flow assay of Salmonella Enteritidis.

In this study, the analytical performance of bacteriophages for Salmonella Enteritidis was investigated using lateral flow assay (LFA) technique. The analytical performance characteristics of bacteriophages were compared with antibodies which are regularly used as analyte-specific agents in the lateral flow immunoassay test strip. Bacteriophages could be an alternative analyte-specific agents to antibodies in lateral flow assay testing of bacteria since they offer comparable sensitivity, specificity, and accuracy. In the present study, Surface Enhanced Raman Spectroscopy (SERS) and colorimetric measurements were combined in one platform and sensitive quantitation of target bacteria was accomplished with a total quantitative analysis time of less than 30 min. The developed Salmonella Enteritidis F5-4 phage-based LFA specifically responds to Salmonella Enteritidis, while lower SERS responses to different bacteria types including Bacillus subtilis, Micrococcus luteus, Escherichia coli, Salmonella Typhimurium were observed. The developed test strips were also applied for the determination of Salmonella Enteritidis in spiked chicken and egg samples.

Development and validation of spectrophotometric and high-performance column liquid chromatographic methods for the simultaneous determination of dienogest and estradiol valerate in pharmaceutical preparations.

Simultaneous determination of dienogest (DIE) and estradiol valerate (EST) in sugar-coated tablets was performed by using HPLC and spectrophotometry. In HPLC, the separation was achieved on an ACE C8 column using the mobile phase acetonitrile-NH4NO3 (0.03 M, pH 5.4; 70 + 30, v/v) at a flow rate of 2 mL/min. The detection wavelength was 280 nm, and cyproterone acetate was selected as an internal standard. The linearity range was 3.0-45.0 microg/mL for DIE and 18.0-100.0 microg/mL for EST. As spectrophotometric methods, two chemometric methods, principal component regression and partial least-squares, were developed. In the chemometric techniques, the concentration data matrix was prepared by using mixtures containing these drugs in methanol-water (3 + 1, v/v). The absorbance data matrix corresponding to the concentration data matrix in these methods was obtained by the measurement of absorbances in their zero-order spectra; then, the calibration was obtained by using the data matrix for the prediction of unknown concentrations of DIE and EST in their binary mixture. Working ranges were found as 2.0-24.0 microg/mL for DIE and 20.0-270.0 microg/mL EST in the methods. These three developed methods were validated and successfully applied to a pharmaceutical preparation, a sugar-coated tablet, and the results were compared with each other.

A Case of Refractory Polymyositis Successfully Treated With Abatacept Monotherapy.

Polymyositis (PM) is an autoimmune disease progressing in the form of a break down of the muscles that is induced by chronic inflammation in skeletal muscles. Muscle weakness is painless and concentrates on proximal muscles, involving the pectoral and pelvic girdle. If the disease is not treated properly, it may progress and lead to a considerable decrease in the quality of life. Its conventional treatment involves drugs that suppress inflammation such as steroids, methotrexate, azathioprine, and intravenous immunoglobulin. However, conventional treatment may prove insufficient to halt the progression of the disease and offer only a limited improvement because of the adverse effects it causes in some patients. In this article, we present a 48-year-old female patient diagnosed with PM nearly 13 years ago that did not sufficiently respond to the pharmaceutical agents that were indicated for the conventional treatment of the disease and developed femoral head avascular necrosis because of the treatment and was, in the end, successfully treated with abatacept monotherapy at our clinic.

Electrochemical, spectroscopic, and molecular docking studies of the interaction between the anti-retroviral drug indinavir and dsDNA.

In this study, an electrochemical DNA biosensor was developed using a straightforward methodology to investigate the interaction of indinavir with calf thymus double-stranded deoxyribonucleic acid (ct-dsDNA) for the first time. The decrease in the oxidation signals of deoxyguanosine (dGuo) and deoxyadenosine (dAdo), measured by differential pulse voltammetry, upon incubation with different concentrations of indinavir can be attributed to the binding mode of indinavir to ct-dsDNA. The currents of the dGuo and dAdo peaks decreased linearly with the concentration of indinavir in the range of 1.0-10.0 µg/mL. The limit of detection and limit of quantification for indinavir were 0.29 and 0.98 µg/mL, respectively, based on the dGuo signal, and 0.23 and 0.78 µg/mL, respectively, based on the dAdo signal. To gain further insights into the interaction mechanism between indinavir and ct-dsDNA, spectroscopic measurements and molecular docking simulations were performed. The binding constant (Kb) between indinavir and ct-dsDNA was calculated to be 1.64 × 108 M-1, based on spectrofluorometric measurements. The obtained results can offer insights into the inhibitory activity of indinavir, which could help to broaden its applications. That is, indinavir can be used to inhibit other mechanisms and/or hallmarks of viral diseases.

Assessment of hand functions in patients with idiopathic cervical dystonia.

Cervical dystonia (CD) is the most common form of focal dystonia characterized by involuntary contractions of the neck muscles, causing abnormal rotation of the head into specific directions. Studies report that idiopathic dystonia is a developmental disorder of the sensorimotor circuits, involving both the cortico-striatal and thalamo-cortical pathways. It is also suggested that enhanced cortical plasticity extends beyond the clinically affected region and may also be detected in the unaffected upper limbs of the patient with CD. In the present study, we aimed at exploring if patients with CD had hand motor dysfunctions. Forty patients with idiopathic CD and 40 healthy controls were included in this study. Dystonic symptoms were assessed by means of The Toronto Western Spasmodic Torticollis Rating Scale (TWSTRS). Stanford Health Assessment Questionnaire (HAQ) was used to assess functional status. Quality of life (QoL) was assessed by using the Medical Outcomes Study Short Form 36-Item Health Survey (SF 36). Grip strength was assessed by using hand dynamometers. Tip pinch, lateral pinch and chuck pinch of the hand were assessed by using a pinchmeter. Fingertip dexterity and hand coordination was assessed using Purdue Pegboard. Duruoz Hand Index (DHI) was used for the assessment of hand functions. There were no significant differences between the groups in grip and pinch strengths of hands and fingers. As to the fingertip dexterity, patients with CD had a mean Pin 1 and Pin 2 test score of 10.6 ± 2.8 and 10.8 ± 3.2 respectively and a mean assembling test score of 5.2 ± 2.0. These results were significantly worse than those of the healthy controls. As to the SF 36 sub-scores, there were significant differences between the groups in all SF 36 sub-scores (p < .001). This study indicates that patients with CD suffer a deteriorated fine motor coordination of hands without dystonic involvement of upper extremities. Furthermore, lower SF 36 scores in patients with CD suggest poorer health-related quality of life.