Novel methods to prepare cross-linked enzyme aggregates (CLEA) challenging immobilization models-urease and pepsin

The common goal of various protein immobilization techniques has been to bypass the intrinsic drawbacks of utilizing free enzymes as catalytic materials in industry. Crosslinked enzyme aggregates (CLEAs), one of the most successful, easily and widely applicable techniques developed so far, has greatly improved the storage and operational stability of enzyme preparations as well as permitted their easy recovery and thus reuse. Involving the seemingly simple semi-specific chemical cross linking of protein aggregates forced out of solution, the general applicability of typical CLEA methods has occasionally been challenged by protein-specific anomalies, reflecting intrinsic structural and functional traits, altering the effectiveness of aggregation and crosslinkability, as well as the resultant bioactivity of the material. In this work, the described limitations, have been addressed using two particularly CLEA-unfriendly protein starting materials, namely, native pepsin and urease. In case of urease, conventional CLEA methods led to dramatically low aggregation and cross linking yields, and displayed statistically insignificant catalytic activity in the immobilized product. Critical breakthrough was achieved by enforcing protein aggregation via lyophilization as opposed to routine precipitation. The subsequent crosslinking of the lyophilizate (yielding a CLEL) in a suitable antisolvent led to a much improved crosslinking yield and catalytic activity. In case of pepsin, the problematic step was achieving covalent crosslinking by conventional CLEA methods, as pepsin bears a single surface lysyl residue and predictably was relatively unresponsive to all crosslinking attempts of surface amino groups. The problem was alleviated by appropriate choice of a rather large crosslinker, i.e., dextran polyaldehyde, and the use of the subzero crosslinking temperatures, therefore permitting the formation of the first ever catalytically competent pepsin CLEA. Novel immobilized formulations presented herein, are expected to contribute as alternatives to many established industrially important applications, involving challenging protein systems. Furthermore, these also could be utilized to prompt greener processes, such as the syntheses of industrially important commodity compounds

The Determination of Agitation Behaviors among the Elderly People Receiving Long-Term Institutional Care and the Influencing Factors

WOS: 000469810700008Amaç: Ajitasyon kurumsal bakım alan yaşlılarda yaygındır. Özellikle demanslı hastalar için önemli bir problemdir. Bu kapsamda çalışmanın amacı bir ilde uzun süreli kurumsal bakım alan yaşlıların ajitasyon davranışlarının ve etkileyen faktörlerle ilişkisinin belirlenmesidir. Yöntem: Araştırma tanımlayıcı ve analitik türdedir. Araştırmanın evrenini, İl Aile ve Sosyal Politikalar Müdürlüğüne bağlı üç kurumsal bakım merkezinde yaşayan yaşlı bireyler oluşturmuştur. Araştırmada örneklem yöntemine gidilmeyip, örneklem grubuna 60 yaş ve üzeri, kendisi ve yakını tarafından çalışmaya katılmasına olur verilen 178 yaşlı birey alınmıştır. Araştırmanın verileri Eylül-Kasım 2018 tarihleri arasında toplanmıştır. Verilerin toplanmasında sosyo-demografik soru formu, Cohen-Mansfield Ajitasyon Envanteri (CMAE), Mini-Cog Kısa Bilişsel Değerlendirme Formu, Barthel Günlük Yaşam Aktiviteleri İndeksi (BGYA), Lawton&Brody Enstrümantal Günlük Yaşam Aktiviteleri Ölçeği (EGYA) kullanılmıştır. Bulgular: Çalışmaya katılan yaşlıların yaş ortalaması 75.58±8.77 olup %56.7’si erkektir. Bulundukları kurumda bakım alma süresi 23.48±24.46 aydır. Yaşlıların %50.6’sının psikiyatrik tanısı bulunmaktadır. Demanslı yaşlıların CMAE puan ortalaması 46.26±15.10 olup demansı olmayanlara göre anlamlı düzeyde yüksektir (Z=-2.132, p=0.033). Mini-Cog puan değerlendirmesine göre bilişsel bozulma olasılığı yüksek olarak belirlenen yaşlıların CMAE puan ortalaması 46.04±15.77 olup düşük bilişsel bozulma olasılığı olanlara göre anlamlı düzeyde yüksektir (Z=-4.137, p=0.000). Sonuç: Çalışmanın sonuçları demanslı hastalarda ajitasyonun yüksek olduğunu, demansla birlikte seyreden bilişsel bozulmanın ajitasyon üzerinde önemli bir etki gösterdiğini ortaya koymuştur.Aim: Agitation is common among elderly people staying at nursing homes. It is a significant problem particularly for patients with dementia. This study aims to determine the agitation behaviors of elderly people receiving long-term institutional care in a city and influencing factors. Method: The population of this descriptive and analytical study consisted of elderly individuals in three institutional care centers. 178 elderly individuals aged 60 and above were taken into the sample group. Approval was taken from them and their relatives. The study data were collected between September-November 2018 using the socio-demographic question form, the Cohen-Mansfield Agitation Inventory (CMAI), the Mini-Cog Brief Cognitive Assessment Form, the Barthel Index for Activities of Daily Living (ADL) and the Lawton&Brody Instrumental Activities of Daily Living Scale (IADL). Results: The mean age of the participants was 75.58 +/- 8.77 and 56.7% were males. The duration of stay at the institution was 23.48 +/- 24.46 months. Although 50.6% of the participants had psychiatric diagnoses. The mean score for the CMAI for the patients with dementia was 46.26 +/- 15.10, which was significantly higher than those without dementia (Z=-2.132, p=0.033). Conclusions: The results show agitation is high among patients with dementia and cognitive impairment affects agitation significantly

Cross-linked enzyme lyophilisates (CLELs) of urease: a new method to immobilize ureases

In this study, we presented a new approach for immobilizing JBU (Jack bean urease), by producing urease cross-linked enzyme lyophilisates (CLELs). Through the use of bovine serum albumin (BSA), lyophilisation, cross-linking with dextran polyaldehyde (DPA), and optimizing cross-linker pH, the urease-CLELs produced show an increase in relative catalytic activity that is 1.47 times higher than that of free urease, while remaining stable up to temperatures of 85 °C. Urease-CLEL activity increases in direct proportion with the increasing BSA content due to the offered additional lysine (Lys) groups which are potential cross-linking points providing better immobilization and retention of JBU, while lyophilisation also enables stabilization by eliminating solvating water molecules and intra-molecular reactions that may block the cross-linking residues. Two most commonly used cross-linkers that are reacting with the available Lys groups, i.e.glutaraldehyde (GA) and bulkier alternative DPA, have been selected for the immobilization of urease. The catalytic activity increase with DPA suggests an improved access to the active site through hindering blockage, while the increase with alkaline pH of the cross-linkers indicates decreased buffer inhibition. The long lifetime (113% residual activity after 4 weeks), recyclability (132% residual activity after 10 cycles) and thermal stability (276% relative activity at 85 °C) of these urease-CLELs demonstrate that they are technologically attractive as green biocatalysts, while our immobilization approach offers an alternative to conventional methods for proteins that are difficult to immobilise

Oxidative chemical vapor deposition (oCVD) synthesis of molecularly imprinted polypyrrole nanotube for the detection of CA-125 protein

Field-effect transistor (FET) based biosensors have been widely used in various applications such as medical diagnosis, health and environmental monitoring. The performance of the FET biosensors are determined by anchoring specific probes on the conducting channel for target biomolecules. In the past two decades, molecularly imprinted polymers (MIPs) have attracted much attention as a robust and cost-effective alternative to natural bioreceptors such as enzymes and antibodies. MIPs are artificial template-made receptors which have the ability to recognise specific target molecules. Here, we present a novel FET biosensor incorporated with a molecularly imprinted polypyrrole nanotube (MIPN) for the selective detection of CA-125 ovarian cancer biomarkers. Polypyrrole (PPy) nanotube used in the FET biosensor has been obtained via oxidative chemical vapor deposition (oCVD) which is a highly efficient solvent-free, vacuum-based technique for the synthesis of conductive polymers. Thanks to the flexibility of this method, MIPN has been conveniently synthesized by coating conformally the sacrificial porous structured templates in the presence of target protein. Particularly, imprinting the target molecule on the nanotube surface has provided extremely high surface area which leads to enhancement in selectivity of the sensor. Furthermore, the oCVD synthesis has enabled higher electrical conductivity for the resulting PPy nanotube by tuning deposition parameters such as oxidant/monomer ratio, substrate temperature and reaction pressure. Synthesized MIPN has been integrated onto the interdigitated array Au electrode to make a conductive bridge between source and drain terminals and biosensor platform has been assembled from chip device, microfluidic channel and custom made chip holder for real-time measurement. The present study has provided useful insight into preparing a novel molecularly imprinted polymer nanotube with various target protein molecules by using oCVD technique

Development of molecularly imprinted polymer-based FET biosensor via oxidative chemical vapor deposition

Biosensors have been attracting an increasing interest in various fields including medical diagnosis and health monitoring due to its rapid, selective and sensitive detection of target analyte in real-time. As biological receptors require special environmental conditions, they limit the durability and long-term storage of the biosensors. However, molecularly imprinted polymers (MIPs) are artificially synthesized to mimic the recognition of biological macromolecules at a significantly lower cost and no need for any special storage. Here, we reported the usability of the polypyrrole (PPy) MIP as a synthetic biorecognition element on a FET based biosensor to detect CA-125 ovarian cancer biomarker. We used oxidative chemical vapor deposition (oCVD) technique which allows the conformal and controlled thin film conductive polymers, to produce MIP onto a sacrificial layer. Synthesized MIP then integrated onto the interdigitated electrode arrays to make a conductive bridge between source and drain terminals for the development of the FET biosensing platform. The oCVD technique has a great potential to be used in the development of MIP-based biosensors for the detection of target proteins without wasting as in the traditional bulk polymerization method

Blends of highly branched and linear poly(arylene ether sulfone)s: multiscale effect of the degree of branching on the morphology and mechanical properties

This study reports the synthesis of highly branched poly(arylene ether sulfone)s (HBPAES) and their incorporation into linear poly(arylene ether sulfone) (LPAES) to investigate the effect of branched topology on the morphological and mechanical properties of final polymer blends. The A2 + B3 polymerization was utilized to synthesize HBPAESs with varying distance between branch points by reacting monomeric 4,4′-dichlorodiphenyl sulfone (DCDPS) or pre-synthesized chlorine terminated linear oligomers with various degrees of polymerization as the A₂ species with 1,1,1-tris(4-hydroxyphenyl)ethane (THPE) as the B₃ monomer. The chemical structure and the degree of branching of synthesized HBPAESs were characterized by 1H Nuclear Magnetic Resonance (NMR) spectroscopy, while Size Exclusion Chromatography (SEC) and Differential Scanning Calorimetry (DSC) were used for the determination of their molecular weight and glass transition temperatures. Polymer blends of HBPAES and LPAES (10/90 w/w) were solution cast into free-standing, dry films and characterized by tensile tests, Dynamic Mechanical Analysis (DMA), Atomic Force (AFM) and Scanning Electron (SEM) Microscopies. Complementary to experimental studies, these blends were modeled with dissipative particle dynamics (DPD) simulations to explain their microphase behavior, miscibility, and morphology. The experimental and computational studies together revealed that understanding the effect of the degree of branching on the intermolecular interactions of highly branched polymers with their linear analogues is critical to obtain final polymer blends with tunable mechanical properties and enhanced fracture behavior