Immobilization of catalase via adsorption on poly(styrene-d-glycidylmethacrylate) grafted and tetraethyldiethylenetriamine ligand attached microbeads

Fibrous poly(styrene-d-glycidylmethacrylate) (P(S-GMA)) brushes were grafted on poly(styrene-divinylbenzene) (P(S-DVB)) beads using surface initiated-atom transfer radical polymerization (SI-ATRP). Tetraethyldiethylenetriamine (TEDETA) ligand was incorporated on P(GMA) block. The multi-modal ligand attached beads were used for reversible immobilization of catalase. The influences of pH, ionic strength and initial catalase concentration on the immobilization capacities of the P(S-DVB)-g-P(S-GMA)-TEDETA beads have been investigated. Catalase adsorption capacity of P(S-DVB-g-P(S-GMA)-TEDETA beads was found to be 40.8 ± 1.7 mg/g beads at pH 6.5 (with an initial catalase concentration 1.0 mg/mL). The K_m value for immobilized catalase on the P(S-DVB-g-P(S-GMA)-TEDETA beads (0.43 ± 0.02 mM) was found about 1.7-fold higher than that of free enzyme (0.25 ± 0.03 mM). Optimum operational temperature and pH was increased upon immobilization. The same support was repeatedly used five times for immobilization of catalase after regeneration without significant loss in adsorption capacity or enzyme activity.     

Reversible immobilization of uricase on conductive polyaniline brushes grafted on polyacrylonitrile film

Polyacrylonitrile film (PAN) surfaces were modified with chemical polymerization of conductive polyaniline (PANI) in the presence of potassium dichromate as an oxidizing agent. The conductive films were used for immobilization of uricase. The surface resistance of the conductive film in this work was found to be 0.97 kΩ/cm. The maximum amount of immobilized enzyme on conductive film containing 2.4% PANI was about 216 μg/cm². The optimum pH for free and immobilized enzymes was observed at 7.0 and 7.5, respectively. The K _m values for free and immobilized uricase were found to be 94 and 138 μM, respectively. V _max values were calculated as 1.87 and 1.63 U/mg protein for the free and immobilized enzymes, respectively. Immobilized uricase exhibited ~68% of its original activity even after 2 months of storage at 4 °C while the free enzyme lost its initial activity within 4 weeks.     

Immobilization of glucoamylase onto polyaniline-grafted magnetic hydrogel via adsorption and adsorption/cross-linking

Glucoamylase (GA) was immobilized onto polyaniline (PANI)-grafted magnetic poly(2-hydroxyethylmethacrylate-co-glycidylmethacrylate) hydrogel (m-p(HEMA-GMA)-PANI) with two different methods (i.e., adsorption and adsorption/cross-linking). The immobilized enzyme preparations were used for the hydrolysis of “starch” dextrin. The amount of enzyme loading on the ferrogel was affected by the medium pH and the initial concentration of enzyme. The maximum loading capacity of the enzyme on the ferrogel was found to be 36.7 mg/g from 2.0 mg/mL enzyme solution at pH 4.0. The adsorbed GA demonstrated higher activity (59%) compared to adsorbed/cross-linked GA (43%). Finally, the immobilized GA preparations exhibited greater stability against heat at 55 °C and pH 4.5 compared to free enzyme (50 °C and pH 5.5), suggesting that the ferrogel was suitable support for immobilization of glucoamylase.     

Mutations in RIPK4 cause the autosomal-recessive form of popliteal pterygium syndrome

The autosomal-recessive form of popliteal pterygium syndrome, also known as Bartsocas-Papas syndrome, is a rare, but frequently lethal disorder characterized by marked popliteal pterygium associated with multiple congenital malformations. Using Affymetrix 250K SNP array genotyping and homozygosity mapping, we mapped this malformation syndrome to chromosomal region 21q22.3. Direct sequencing of RIPK4 (receptor-interacting serine/threonine kinase protein 4) showed a homozygous transversion (c.362T>A) that causes substitution of a conserved isoleucine with asparagine at amino acid position 121 (p.Ile121Asn) in the serine/threonine kinase domain of the protein. Additional pathogenic mutations-a homozygous transition (c.551C>T) that leads to a missense substitution (p.Thr184Ile) at a conserved position and a homozygous one base-pair insertion mutation (c.777_778insA) predicted to lead to a premature stop codon (p.Arg260ThrfsX14) within the kinase domain-were observed in two families. Molecular modeling of the kinase domain showed that both the Ile121 and Thr184 positions are critical for the protein's stability and kinase activity. Luciferase reporter assays also demonstrated that these mutations are critical for the catalytic activity of RIPK4. RIPK4 mediates activation of the nuclear factor-κB (NF-κB) signaling pathway and is required for keratinocyte differentiation and craniofacial and limb development. The phenotype of Ripk4(-/-) mice is consistent with the human phenotype presented herein. Additionally, the spectrum of malformations observed in the presented families is similar, but less severe than the conserved helix-loop-helix ubiquitous kinase (CHUK)-deficient human fetus phenotype; known as Cocoon syndrome; this similarity indicates that RIPK4 and CHUK might function via closely related pathways to promote keratinocyte differentiation and epithelial growth.     

Effect of cadmium on antioxidant status in alloxane-induced diabetic rats

Fifty-two healthy Swiss Male Albino rats aged two mo were used in this study. They were divided into four groups: control (C), diabetic (D), cadmium (Cd), and diabetic+Cd (D+Cd) groups. Diabetic condition was induced in D and D+Cd groups by administration of alloxane (5 mg/100 g). After this treatment, CD and D+Cd groups were injected with CdCl₂ ip (2 mg/kg/wk). At the end of the 2-mo experimental period, thiobarbituric acid reactive substances (TBARS), plasma and erythrocyte selenium (SE), plasma ceruloplasmin (Cp), and vitamin E (vit E) were determined in four groups of rats. The erythrocyte Se was lower in the experimental groups than in the controls. Plasma Se was significantly decreased in the D and D+Cd groups compared with the control group. Plasma Cp was unaltered. Plasma vit E was significantly decreased in Cd group in comparison with the C, D, and D+Cd groups.     

Evaluation of method performance for oxidative stress biomarkers in urine and biological variations in urine of patients with type 2 diabetes mellitus and diabetic nephropathy

Background

Oxidative stress biomarkers such as superoxide dismutase (CuZnSOD), catalase (CAT) and malondialdehyde (MDA) play an important role in the pathogenesis or progression of numerous diseases. Data regarding the biological variation and analytical quality specifications (imprecision, bias and total error) for judging the acceptability of method performance for oxidative stress biomarkers in urine are conspicuously lacking in the literature. Such data are important in setting analytical quality specifications, assessing the utility of population reference intervals (index of individuality) and assessing the significance of changes in serial results from an individual (reference change value; RCV).

Materials and methods

20 patients with type 2 diabetes mellitus (T2DM), 20 patients with diabetic nephropathy (DN) and 14 healthy individuals as control were involved in this study. Timed first morning urine samples were taken from patients and healthy groups on the zero, 1st, 3rd, 5th, 7th, 15th and 30th days. Index of individuality and reference change value were calculated from within-subject and between-subject variations. Methods of oxidative stress biomarkers in human blood were adopted in human urine and markers were measured as spectrophotometrically. Also, analytical quality specifications for evaluation of the method performance were established for oxidative stress biomarkers in urine.

Results

Within-subject variations of oxidative stress biomarkers were significantly higher in patients with DN and T2DM compared to healthy subjects. MDA showed low individuality, and within-subject variances of MDA were larger than between-subject variances in all groups. However, CAT and CuZnSOD showed strong individuality, but within-subject variances of them were smaller than between-subject variances in all groups. RCVs of all analytes in diabetic patients were relatively higher, because of high within-subject variation, resulting in a higher RCV. Also, the described methodology achieves these goals, with analytical CVs of < 3.5% for all analytes. Goals for bias and total error were 6.0-7.9% and 12.5-23.3%, respectively.

Conclusions

RCVs concept for predicting the clinical status in diabetic patients represents an optimization of laboratory reporting and could be a valuable tool for clinical decision. Furthermore, for oxidative stress biomarkers’ measurements in urine, the desirable imprecision goals based on biological variation are obtainable by current methodologies.     

Immobilization of catalase via adsorption onto -histidine grafted functional pHEMA based membrane

Poly(2-hydroxyethylmethacrylate) (pHEMA) based flat sheet membrane was prepared by UV-initiated photopolymerization technique. The membrane was then grafted with -histidine. Catalase immobilization onto the membrane from aqueous solutions containing different amounts of catalase at different pH was investigated in a batch system. The maximum catalase immobilization capacity of the pHEMA–histidine membrane was 86 μg cm⁻². The activity yield was decreased with the increase of the enzyme loading. It was observed that there was a significant change between V_max value of the free catalase and V_max value of the adsorbed catalase on the pHEMA–histidine membrane. The K_m value of the immobilized enzyme was higher 1.5 times than that of the free enzyme. Optimum operational temperature was 5°C higher than that of the free enzyme and was significantly broader. It was observed that enzyme could be repeatedly adsorbed and desorbed without loss of adsorption capacity or enzyme activity.     

Targeting CoV-2 spike RBD and ACE-2 interaction with flavonoids of Anatolian propolis by in silico and in vitro studies in terms of possible COVID-19 therapeutics

Propolis is a multi-functional bee product rich in polyphenols. In this study, the inhibitory effect of Anatolian propolis against SARS-coronavirus-2 (SARS-CoV-2) was investigated in vitro and in silico. Raw and commercial propolis samples were used, and both samples were found to be rich in caffeic acid, p-coumaric acid, ferulic acid, t-cinnamic acid, hesperetin, chrysin, pinocembrin, and caffeic acid phenethyl ester (CAPE) at HPLC-UV analysis. Ethanolic propolis extracts (EPE) were used in the ELISA screening test against the spike S1 protein (SARS-CoV-2): ACE-2 interaction for in vitro study. The binding energy values of these polyphenols to the SARS-CoV-2 spike and ACE-2 protein were calculated separately with a molecular docking study using the AutoDock 4.2.6 program. In addition, the pharmacokinetics and drug-likeness properties of these eight polyphenols were calculated according to the SwissADME tool. The binding energy value of pinocembrin was highest in both receptors, followed by chrysin, CAPE, and hesperetin. Based on the in silico modeling and ADME (absorption, distribution, metabolism, and excretion) behaviors of the eight polyphenols, the compounds exhibited the potential ability to act effectively as novel drugs. The findings of both studies showed that propolis has a high inhibitory potential against the Covid-19 virus. However, further studies are now needed.     

Reversible immobilization of Candida rugosa lipase on fibrous polymer grafted and sulfonated p(HEMA/EGDMA) beads

Poly(2-hydroxyethyl methacrylate/ethylenglycol dimethacrylate) beads were grafted with poly(glycidylmethacrylate) via surface initiated atom transfer radical polymerization. Epoxy groups of the grafted polymer were modified in to sulfone groups. Sulfonated beads were characterized by swelling studies, FT-IR, SEM and elemental analysis, and were used for reversible immobilization of lipase. Under given experimental conditions, the beads had an adsorption capacity of 44.7 mg protein/g beads. The adsorbed lipase on beads retained up to 67.4% of its initial activity. The immobilized lipase exhibited improved thermal and storage stabilities over those of the free enzyme. The immobilized lipase could desorb 1.0 M NaCl solution at pH 8.0, and the sulfonated beads can be repeatedly charged with fresh enzyme after inactivation upon use.     

P(HPMA/EGDMA) beads grafted with fibrous chains by SI-ATRP method: agmatine functionalized affinity beads for selective separation of serum albumin

In this paper, novel core–shell polymeric affinity beads based on fibrous grafting and functionalization with a salt resistance affinity ligand were developed to separate and deplete serum albumin (SA) from human serum. Poly(hydroxypropyl methacrylate/ethyleneglycole dimethacrylate), p(HPMA/EGDMA), beads were prepared via suspension polymerization, and were grafted with poly(glycidyl methacrylate) (p(GMA)) via surface-initiated atom transfer radical polymerization (SI-ATRP) method. The grafted p(GMA) fibrous chains on the beads were modified with an affinity ligand (i.e., agmatine). The binding capacity of the affinity beads to SA was determined using aqueous solution of SA in a batch system. Batch adsorption studies showed that the amount of adsorbed SA was found to be 156.7 mg/g at 25 °C. The maximum adsorption capacity for affinity beads was observed at around pH 5.5. Adsorption of SA onto affinity beads significantly increased with increasing temperature, and reached a value 177.8 mg/g beads at 35 °C. The equilibrium data were found to be well described by Langmuir model, while the kinetic data were well fitted to the pseudo-second-order kinetic. The degree of the purity of SA was determined by using HPLC. Before and after adsorption, the peak areas of SA were used in the calculation of separated SA.