New Sintered Porous Scaffolds of Mg,Sr Co-Substituted Hydroxyapatite Support Growth and Differentiation of Primary Human Osteoblasts In Vitro

Strontium (Sr) and Magnesium (Mg) are bioactive ions that have been proven to exert a beneficial effect on bone; therefore, their incorporation into bone substitutes has long been viewed as a possible approach to improve tissue integration. However, the thermal instability of Mg-substituted hydroxyapatites has hitherto limited development. We previously described the creation of thermally consolidated porous constructs of Mg,Sr co-substituted apatites with adequate mechanical properties for their clinical use. The present paper describes the biocompatibility of Mg,Sr co-substituted granules using an alveolar-bone-derived primary model of human osteoblasts. Cells were cultured in the presence of different amounts of hydroxyapatite (HA), Sr-substituted HA, or MgSrHA porous macrogranules (with a size of 400–600 microns, obtained by grinding and sieving the sintered scaffolds) for three and seven days, and their viability was measured by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Protein content was measured using the Lowry assay at the same time points. Cell viability was not impaired by any of the tested compounds. Indirect and direct biocompatibility of these macrogranules was assessed by culturing cells in a previously conditioned medium with HA, SrHA, or MgSrHA, or in the presence of material granules. Osteoblasts formed larger and more numerous nodules around SrHA or MgSrHA granules. Furthermore, cell differentiation was evaluated by alkaline phosphatase staining of primary cells cultured in the presence of HA, SrHA, or MgSrHA granules, confirming the increased osteoconductivity of the doped materials.

Ionic gold demonstrates antimicrobial activity against Pseudomonas aeruginosa strains due to cellular ultrastructure damage

Due to the ever-increasing rise of antimicrobial resistant (AMR) bacteria, the development of alternative antimicrobial agents is a global priority. The antimicrobial activity of ionic gold was explored against four Pseudomonas aeruginosa strains with different AMR profiles in order to determine the antimicrobial activity of ionic gold and elucidate the mechanisms of action. Disc diffusion assays (zone of inhibition: ZoI) coupled with minimum inhibitory/bactericidal concentrations (MIC/MBC) were conducted to determine the antimicrobial efficacy of ionic gold. Scanning electron microscopy (SEM) was used to visualise morphological changes to the bacterial cell ultrastructure. Strains with increased AMR were slower to grow which is likely a fitness cost due to the enhanced AMR activity. Although greater concentrations of ionic gold were required to promote antimicrobial activity, ionic gold demonstrated similar antimicrobial values against all strains tested. Lowry assay results indicated that protein leakage was apparent following incubation with ionic gold, whilst SEM revealed cellular ultrastructure damage. This study suggests that the application of ionic gold as an alternative antimicrobial is promising, particularly against AMR P. aeruginosa. The antimicrobial activity of ionic gold against P. aeruginosa could potentially be utilised as an alternative therapeutic option in wound management, an approach that could benefit healthcare systems worldwide.

Estimation of the Acetylcholinesterase activity of honey bees in Nepal

Decline in honey bee colonies possess a serious threat to biodiversity and agriculture. Prior detection of the stresses with the help of biomarkers and their management ensures honey bee’s survivability. Acetylcholinesterase (AChE) is a promising biomarker to monitor exposure of honey bees towards environmental pollutants. In this preliminary study, we measured AChE activity in forager honey bees collected from six districts of Nepal, Kathmandu, Bhaktapur, Lalitpur, Chitwan, Rupandehi and Pyuthan during autumn and winter seasons. We estimated AChE tissue and specific activities from bee’s heads using commercial kit based on Ellman assay and protein concentration using Lowry assay. In total, we collected 716 foragers belonging to A. cerana, A. mellifera and A. dorsata. A significant increase in all three parameters measured: AChE tissue activity, AChE specific activity and protein concentration was observed in winter samples. Both AChE tissue and specific activities were lower in A. mellifera compared to either A. cerana or A. dorsata. Protein concentration was higher in A. mellifera than in A. dorsata and lower than in A. cerana. We show correlation between both AChE tissue and specific activities and protein concentration across season and species and discuss possible factors contributing to the observations. Our results clearly indicate the presence of stress in the winter which is manifested through overexpression of the AChE. We recommend a detailed study to determine the factors accountable for the stresses for better management of honey bees in Nepal.

Quantification of Soluble or Insoluble Fractions of Leishmania Parasite Proteins in Microvolume Applications: A Simplification to Standard Lowry Assay

Protein quantification is often an essential step in any research field that involves proteins. Although the standard Lowry assay and its modifications are most abundantly used in protein quantification, the existing methods are rigid or often demonstrate nonlinearity between protein concentration and color intensity. A method for fast and accurate qualitative and/or quantitative determination of total soluble/insoluble proteins or micro-well plate immobilized proteins isolated from Leishmania parasites in microvolumes was described in the current study. Improvements in cost-effective techniques are necessary to increase the research outputs in resource-limited settings. This method is a modification to the established Lowry assay for protein quantification. Concentrations of unknown samples were calculated using a standard curve prepared using a standard series of bovine serum albumin (BSA). The optimized reagents were 2 N NaOH (sodium hydroxide), 2% Na2CO3 (sodium carbonate), 1% CuSO4 (copper sulfate), 2% KNaC4H4O6 (potassium sodium tartrate), and 2 N Folin and Ciocalteu’s phenol. This modified protein assay was sensitive for quantifying Leishmania proteins in a total crude extract or in a soluble fraction within the approximate range of 10–500 μg/ml (1–50 μg/assay) and showed a linearity between color intensity and concentration of the protein. This is an easier, fast, and accurate method for quantifying proteins with microvolumes in a cost-effective manner for routine use in research laboratories in resource-limited settings.

Preliminary Assessment of the Antihypertensive and Antioxidative Activities of the Peptides from “Saba” Banana (Musa balbisiana Colla) Flesh

The crude proteins were isolated using 0.125 M Tris-HCl with 50 mM NaCl at pH 7.4. The protein content of the crude extract was determined using the Lowry assay and was found to be 4.28 mg/mL. The major band which corresponds to the major protein has an approximate molecular weight of 20 KDa. The isolated crude proteins were subjected to enzymatic digestion using pepsin, trypsin, chymotrypsin, and thermolysin for 3, 4, 12, and 24 hours. The 24-hour digest was found to have the highest percent anti-Angiotensin Converting Enzyme (ACE) activity (36.02%) while the 12-hour digest was found to have the highest anti-oxidative activity (33.14%) using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The 24-hour digest was subjected to Ultra-Performance Liquid Chromatography (UPLC) to determine the peptide fraction responsible for the ACE inhibitory activity. Three peptide fractions (PF1, PF2 and PF3) were chosen and PF2 exhibited the highest percent inhibitory activity (32.21) against ACE. PF2 was subjected to thin layer chromatography (TLC) and the possible identity is EK based on the Rf value traveled by glutamic acid (E) (0.43) and Lysine (K) (0.13). In silico analysis was done to correlate the results with the presence of putative peptides with antioxidative and antihypertensive activities. Results showed that antihypertensive peptides EK, GS, TY, FNE, FP, LKA, PT, PP, FAL and antioxidative peptides IR and VPW were found based on the sequence of the protein in “Saba” banana. The presence of the antihypertensive peptide EK was verified using thin layer chromatography (TLC).

The Microwave-assisted Synthesis of Polyethersulfone (PES) as A Matrix in Immobilization of Candida antarctica Lipase B (Cal-B)

Candida antarctica lipase B (Cal-B) has been widely used in the hydrolysis reaction. However, it has some weaknesses, such as: forming of the heavy emulsion during the process, which is difficult to resolve and has no reusability. Therefore, it needs to be immobilized into a suitable matrix. One of the suitable supporting materials is polyethersulfone (PES) and its synthesis becames the objective of this paper. The PES was synthesized via a polycondensation reaction between hydroquinone and 4,4'-dichlorodiphenylsulfonein N-methylpyrrolidone (NMP) as solvent using Microwave Assisted Organic Synthesis (MAOS) method at170 °C for 66 minutes using an irradiation power of 300 watt. The synthesized PES was characterized by FTIR and 1H-NMR (500 MHz, DMSO-d6). Then the PES membrane was prepared from 20 % of the optimized mixtures of PES, PSf (polysulfone), and PEG (polyethylene glycol) dissolved in 80 % NMP. The Cal-B was immobilized on the PES membrane by mixing it in a shaker at 30 °C and 100 rpm for 24 h using phosphate buffered saline (PBS). The identification of the immobilized Cal-B was done by using FTIR-ATR spectroscopy and SEM micrographs. The results of Lowry assay showed that the ‘Cal-B immobilized’ blended-membrane has a loading capacity of 91 mg/cm2 in a membrane surface area of 17.34 cm2. In this work, the activity of immobilized Cal-B was twice higher than the native enzyme in p-NP (p-Nitrophenolpalmitate) hydrolyzing. The results indicated that the synthesized PES showed a good performance when used as a matrix in the immobilization of Cal-B. Copyright © 2017 BCREC Group. All rights reservedReceived: 15th November 2016; Revised: 27th May 2017; Accepted: 24th May 2017; Available online: 23rd October 2017; Published regularly: December 2017How to Cite: Widhyahrini, K., Handayani, N., Wahyuningrum, D., Nurbaiti, S., Radiman, C.L. (2017). The Microwave-assisted Synthesis of Polyethersulfone (PES) as A Matrix in Immobilization of Candida antarctica Lipase B (Cal-B). Bulletin of Chemical Reaction Engineering & Catalysis, 12(3): 343-350 (doi:10.9767/bcrec.12.3.774.343-350)

Development of an Economical Method to Reduce the Extractable Latex Protein Levels in Finished Dipped Rubber Products

Natural rubber latex (NRL) allergy is caused by the extractable latex proteins in dipped rubber products. It is a major concern for the consumers who are sensitive to the allergenic extractable proteins (EP) in products such as NRL gloves. Objective of this research was to develop an economical method to reduce the EP in finished dipped NRL products. In order to reduce the EP levels, two natural proteases, bromelain from pineapple and papain from papaya, were extracted and partially purified using (NH4)2SO4. According to the newly developed method, different glove samples were treated with a 5% solution of each partially purified enzyme, for 2 hours at 60°C. Residual amounts of in treated samples were quantified using the modified Lowry assay (ASTM D5712-10). Bromelain displayed a 54 (±11)% reduction of the EP from the dipped rubber products, whereas it was 58 (±8)% with papain. These results clearly indicate that the selected natural proteases, bromelain, and papain contribute significantly towards the reduction of the total EP in finished NRL products. Application of bromelain enzyme for the aforementioned purpose has not been reported up to date, whereas papain has been used to treat raw NRL towards reducing the EP.

Development of a continuous protein and polysaccharide measurement method by Sequential Injection Analysis for application in membrane bioreactor systems

Polysaccharides and proteins are the main components of extracellular polymeric substances, which are considered to give a major contribution to the overall fouling potential of the MBR system. This work focuses on the automation of spectrophotometrical assays for protein and polysaccharide determination for on-line measurement in MBR. Sequential Injection Analysis (SIA) was identified as more suitable for continuous measurements. In screening tests the selection of an appropriate assay for the successful automation by means of SIA was carried out. Lowry Assay and Dubois Assay were chosen for protein and polysaccharide determination. Lowry Assay could be successfully automated by means of SIA. The automated method shows a lower sensitivity than the manual Lowry method, what can be contributed to specific characteristic of SIA. The confidence limit was calculated to 2.8 mg/L. The automated method shows in validation tests good correspondence with protein concentrations measured with the manual assay (R2=0.971). The successful establishment of the Dubois Assay for polysaccharides failed so far due to sensitivity problems.