Optimization of non-detergent treatment for enveloped virus inactivation using the Taguchi design of experimental methodology (DOE)

Abstract

In mammalian cell culture technology, viral contamination is one of the main challenges; and, so far, various strategies have been taken to remove or inactivate viruses in the cell-line production process. The suitability and feasibility of each method are determined by different factors including effectiveness in target virus inactivation, maintaining recombinant protein stability, easiness—in terms of the process condition, cost-effectiveness, and eco-friendliness. In this research, Taguchi design-of-experiments (DOE) methodology was used to optimize a non-detergent viral inactivation method via considering four factors of temperature, time, pH, and alcohol concentration in an unbiased (orthogonal) fashion with low influence of nuisance factors. Herpes Simplex Virus-1 (HSV1) and Vero cell-line were used as models for enveloped viruses and cell-line, respectively. Examining the cytopathic effects (CPE) in different dilutions showed that pH (4), alcohol (15%), time (120?min), and temperature (25?°C) were the optimal points for viral inactivation. Evaluating the significance of each parameter in the HSV-1 inactivation using Taguchi and ANOVA analyses, the contributions of pH, alcohol, temperature and time were 56.5%, 19.2%, 12%, and 12%, respectively. Examining the impact of the optimal viral treatment condition on the stability of model recombinant protein-recombinant human erythropoietin, no destabilization was detected.     

Effect of Soil Fabric on Transport of a LNAPL through Unsaturated Fine-Grained Soils: A Centrifugal Model Study

Centrifugal model tests were performed to study the impact of the fabric of a fine-grained soil on transport of a light non-aqueous phase liquid (LNAPL). An image processing technique was developed to extract contaminant transport and fate data from the centrifugal model. Two unconsolidated sites with different moisture contents and a saturated site consolidated due to self-weight were simulated using the centrifuge. The LNAPL migrated in the vertical direction as a narrow plume and formed a free product pool above the saturated zone in unsaturated and unconsolidated soils. However, the LNAPL migrated in the horizontal direction before moving in the vertical direction as a broad plume in the consolidated site. The test results showed that the final width of the plume in the unsaturated zone of the consolidated site was nearly two times as large as that for the unconsolidated sites. In addition, the rate of leak from the underground storage tanks (USTs) on consolidated soils was substantially higher when compared with those on the unconsolidated state. The comparison of LNAPL saturation profiles at the centerline of the centrifugal models during leakage showed that, depending on the soil fabric at a given time and depth, the LNAPL phase would be different; i.e., mobile or immobile (residual) in the same soil type. The test results provided additional insight into contribution of soil fabric on transport and fate of contaminants. The soil fabric controls the geological and hydro-geological properties of fine-grained soils and hence the contamination plume.     

Feature ranking based on synergy networks to identify prognostic markers in DPT-1

Interaction among different risk factors plays an important role in the development and progress of complex disease, such as diabetes. However, traditional epidemiological methods often focus on analyzing individual or a few ‘essential’ risk factors, hopefully to obtain some insights into the etiology of complex disease. In this paper, we propose a systematic framework for risk factor analysis based on a synergy network, which enables better identification of potential risk factors that may serve as prognostic markers for complex disease. A spectral approximate algorithm is derived to solve this network optimization problem, which leads to a new network-based feature ranking method that improves the traditional feature ranking by taking into account the pairwise synergistic interactions among risk factors in addition to their individual predictive power. We first evaluate the performance of our method based on simulated datasets, and then, we use our method to study immunologic and metabolic indices based on the Diabetes Prevention Trial-Type 1 (DPT-1) study that may provide prognostic and diagnostic information regarding the development of type 1 diabetes. The performance comparison based on both simulated and DPT-1 datasets demonstrates that our network-based ranking method provides prognostic markers with higher predictive power than traditional analysis based on individual factors.     

1,2 Propanediol utilization by Lactobacillus reuteri DSM 20016, role in bioconversion of glycerol to 1,3 propanediol, 3-hydroxypropionaldehyde and 3-hydroxypropionic acid

The objective of the presented work is to demonstrate the metabolism of 1,2 propandiol by Lactobacillus reuteri and to elucidate the metabolites produced during the process. This Metabolic pathway is crucial for biotechnological applications using L. reuteri in bioconversion of glycerol to industrially important plate-form chemicals. L. reuteri grown on minimal media containing 1,2 propanediol was able to utilize the compound as a sole carbon and energy source. The growth of the bacteria was linear with time; however the specific growth rate was significantly low compared to bacteria grown on the same media in the presence of glucose.The fermentation of 1,2 propanediol by L. reuteri in presence and absence of glucose was followed for 72 h and the metabolites produced during the process were detected using HPLC. 1,2 Propanediol was completely converted to propionaldhyde in a time dependent fashion, this process had a higher rate in presence of glucose. Consequently the produced propionaldhyde was converted to propionic acid and propanol in a skewed equimolar manner. In presence of glucose: acetic acid, lactic acid, succinic acid and ethanol were detected while in absence of glucose only minute amounts of acetic acid and lactic acid were detected which indicates presence of different metabolic pathways for glucose and 1,2 propanediol metabolism. Resting cells of L. reuteri induced in presence of 1,2 propanediol have shown significant capabilities to convert aqueous glycerol to 1,3 propanediol, 3-hydroxypropionaldhyde and a compound proposed to be 3-hydroxypropionic acid as detected by gas chromatographic technique.     

Determination of naphazoline hydrochloride in biological and pharmaceutical samples by a quantum dot‐assisted chemiluminescence system using response‐surface methodology

A simple and highly sensitive chemiluminescence (CL) method is reported for the determination of naphazoline hydrochloride (NH). It was found that the weak CL from the reaction of luminol and KIO₄ in an alkaline medium could be highly amplified by cysteine‐capped cadmium telluride quantum dots (QDs) and the enhanced CL was effectively quenched by NH and this finding was utilized as a basis for the determination of NH. The QDs were synthesized in aqueous medium and characterized by X‐ray diffraction (XRD), transmission electron microscopy (TEM), and UV‐vis and photoluminescence spectroscopy. A possible mechanism was proposed for the CL system based on radical identification experiments, along with CL spectrum of the system. The experimental parameters were optimized by the reliable response surface methodology (RSM). Under the optimized experimental conditions, the proposed method allowed the determination of NH over the range of 5.0 × 10^‐10–2.0 × 10^‐7 mol/L (r² = 0.9993, n = 10). The precision (RSD%) of the method, obtained from five replicate determinations of 2.0 and 150 nmol/L NH, was found to be 1.0% and 1.3%, respectively. The method was successfully applied to the determination of NH in pharmaceutical formulations and human urine and serum samples with results corroborated with the aid of those obtained from a standard method. Copyright © 2014 John Wiley & Sons, Ltd.     

Study of Peptide Mimetics of Hepatitis A Virus Conjugated to Keyhole Limpet Hemocyanin and as Multiple Antigen Peptide System

Mimotopes mimic binding properties of natural antigen epitopes. They could be used for vaccine design, drugs development, and diagnostic assays. We have previously identified four bacteriophages displaying hepatitis A virus (HAV) mimotopes from a phage-display peptide library by affinity selection on serum antibodies from hepatitis A patients. Three of these HAV mimotopes showed similarity in their amino acid sequences with at least one of the VP3 and VP1 antigenic proteins of HAV and the four induced specific anti-HAV antibodies. In the present work, four conjugations were done. In each of them, a linear peptide (46, 53, 54 or 56) containing the amino-acid sequence of the corresponding mimotope was conjugated to keyhole limpet hemocyanin (KLH). Conjugation products were named: 46KLH, 53KLH, 54KLH and 56KLH. A two-arm multiple antigen peptide (MAP) system containing peptide sequence 46, and a second MAP containing two copies of peptide sequence 56 were synthesized and dimerized, to obtain the heterodimeric four-arms MAP (named MAP46-56) containing two copies of peptides 46 and 56. Mice were immunized with peptides conjugated to KLH and MAP46-56 to evaluate the ability of these two forms of mimotope presentation, to elicit antibodies that bind to the original antigen. KLH conjugated peptides rendered the highest levels of anti-peptide antibodies and were the only ones that induced specific anti-HAV antibodies. The results of immunizations showed that for the mimotopes chosen here, conjugation to a carrier protein was the most effective option to induce antibodies that cross-reacted with the natural antigen.     

Delineation of temperature-humidity index (THI) as indicator of heat stress in riverine buffaloes (Bubalus bubalis) of a sub-tropical Indian region

The erstwhile developed temperature-humidity index (THI) has been popularly used to indicate heat stress in dairy cattle and often in buffaloes. However, scientific literature suggests differences in thermotolerance and physiological responses to heat stress between cattle and buffalo. Therefore, THI range used to indicate degree of heat stress (mild, moderate, and severe) in cattle should be recalibrated for indicating heat stress in buffaloes. The present study was carried out to delineate THI range to indicate onset and severity of heat stress in buffaloes based on physiological, biochemical, and expression profiling of heat shock response (HSR) genes in animals at different THI. The result indicated early onset of heat stress in buffaloes as compared to cattle. Physiological and biochemical parameters indicated onset of mild signs of heat stress in buffaloes at THI 68-69. Significant deviation in these parameters was again observed at THI range 73-76. At THI 77-80, the physiological and biochemical responses of animals were further intensified indicating extreme alteration in homeostasis. The in vivo expression profiling of HSR genes indicated that members of Hsp70 gene family are expressed in a temporal pattern over different THIs, whereas expressions of Hsf genes were evident during intense heat stress. Overall, the study established that amplitude of heat shock response and THI range for indicating severity of thermal stress for buffaloes are not in unison to cattle. The study also suggests skin temperature of the poll region could be used as non-invasive tool for monitoring heat stress in dairy buffaloes.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12192-021-01209-1.