The Conformational Structural Change of Soy Glycinin via Lactic Acid Bacteria Fermentation Reduced Immunoglobulin E Reactivity

This study investigated the fermentation of isolated soy glycinin by using the Lactiplantibacillus plantarum B1-6 strain, its reduction effect on immunoglobulin E (IgE) reactivity, the relationship with protein aggregation/gelation state and conformational changes. Fermentation was performed under different glycinin concentrations (0.1%, 0.5%, 1% and 2%, w/v) and varied fermentation terminal pH levels (FT-pH) (pH 6.0, 4.5, 4.0 and 3.5). L. plantarum B1-6 showed potency in reducing immunoreactivity to 0.10–69.85%, as determined by a sandwich enzyme-linked immunosorbent assay. At a FT-pH of 6.0 and 4.5, extremely low IgE reactivity (0.1–22.32%) was observed. Fermentation resulted in a great increase (2.31–6.8-fold) in particle size and a loss of intensity in A3 and basic subunits. The conformation of glycinin was altered, as demonstrated by improved surface hydrophobicity (1.33–7.39-fold), decreased intrinsic fluorescence intensity and the α-helix structure. Among the four selected concentrations, glycinin at 1% (w/v, G-1) evolved the greatest particles during fermentation and demonstrated the lowest immunoreactivity. Principal component analysis confirmed that particle size, intrinsic fluorescence intensity, α-helix and ionic bond were closely related to immunoreactivity reduction.

A new replication medium enables a rapid identification of φx-174 virus by synchronous fluorescence of Tryptophan.

Development of rapid methods for identification of bacteriophages based on their intrinsic fluorescence is challenging. Pure bacteriophages may be detected based on the strong fluorescence of the amino acid Tryptophan that exist in their proteins. Nevertheless, Tryptophan is a molecule that also exist in high quantities in the bacterial hosts and their cultivation media. In this work, we show that simple separation of the bacteriophage φx-174 from its E.coli host (grown on standard cultivation medium) by filtration is not sufficient for its identification based on the intrinsic fluorescence of its Tryptophan content. This is mostly because of the tryptophan residues that derive from the cultivation medium. We fabricate a new cultivation medium that does not have any significant fluorescence overlap with Tryptophan. By utilization of this new cultivation medium, we can identify φx174 based on the spectral fingerprint of its intrinsic Tryptophan content by synchronous fluorescence measurements.

Intrinsic fluorescence–based label-free detection of bovine amyloid A amyloidosis

Amyloidosis is diagnosed by the histologic detection of amyloid deposits; however, this method has limitations such as a prolonged diagnosis time and the need for histologic proficiency. We aimed to develop a rapid and simple method for diagnosing amyloidosis by targeting amyloid-specific endogenous fluorescence, which has not been reported previously, to our knowledge. Fluorescence fingerprint analysis of amyloid extracts and tissue homogenates derived from amyloid A (AA) amyloidosis-affected cattle exhibited a specific intrinsic fluorescence pattern. Furthermore, principal component analysis using analytical data revealed that AA could be identified by peaks near λex 350 nm and λem 430 nm. Fluorescence spectrometry analysis using tissue homogenates, which does not require special histochemical staining, enables the rapid detection of bovine AA.

Epigenetics of skin intrinsic florescence

Introduction & Objective: Cumulated advanced glycation end products (AGEs) in the bloodstream and tissues contribute to the pathogenesis of diabetes complications. The skin intrinsic fluorescence (SIF) is a non-invasive measurement of dermal AGEs level using spectrometer, and it can be used as a biomarker in AGEs-related diseases. Previously, specific epigenomic factor has been found to be associated with haemoglobin A1c (HbA1c). HbA1c is a type of glycated haemoglobin – the HbA1c test measures the average glycemic control over the period of 3 months. However, the effect of epigenetic factors on the level of AGEs in the skin remains unknown. We hypothesize that some cytosine-guanine dinucleotides (CpGs) are associated with SIF. An epigenome-wide associations study (EWAS) was performed to identify CpG sites associated with SIF in people with type 1 diabetes. Methods: 499 people with type 1 diabetes that have both methylation and SIF from the Diabetes Control Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) study were included. We fit linear regression models for SIF with each CpG site one at a time. The epigenome-wide significance level (p=5e-8) was applied. Then the result is compared with the null hypothesis where CpGs are not associated with SIF to check the inflation. In order to check the assumptions of the multiple linear models at a single CpG, we use diagnostic plots. Results: We did not identify a specific CpG that is significantly associated with neither skin intrinsic fluorescence 1 (SIF1) nor skin intrinsic fluorescence 12 (SIF 12).The CpG site with strongest effect is cg06538183 ([SE] -2.73 [0.61], p = 8.72e-6) on SIF1 and cg12871967 ([SE] 2.52, 0.53, p = 2.71e-6) on SIF12. Conclusion: We did not find any specific CpG that was significantly associated with either SIF 1 or SIF12. In general, the result suggests that DNA methylation does not impact the accumulation of AGEs in skin cells. DNA methylation data has a unique pattern of distribution that drives the non-uniform distribution of the p-values. The group of 275,301 CpGs that have means above the median and standard deviations below the median has the expected uniform p-value distribution.

Fluorescence Spectroscopic Analysis of ppGpp Binding to cAMP Receptor Protein and Histone-Like Nucleoid Structuring Protein

The cyclic AMP receptor protein (CRP) is one of the best-known transcription factors, regulating about 400 genes. The histone-like nucleoid structuring protein (H-NS) is one of the nucleoid-forming proteins and is responsible for DNA packaging and gene repression in prokaryotes. In this study, the binding of ppGpp to CRP and H-NS was determined by fluorescence spectroscopy. CRP from Escherichia coli exhibited intrinsic fluorescence at 341 nm when excited at 280 nm. The fluorescence intensity decreased in the presence of ppGpp. The dissociation constant of 35 ± 3 µM suggests that ppGpp binds to CRP with a similar affinity to cAMP. H-NS also shows intrinsic fluorescence at 329 nm. The fluorescence intensity was decreased by various ligands and the calculated dissociation constant for ppGpp was 80 ± 11 µM, which suggests that the binding site was occupied fully by ppGpp under starvation conditions. This study suggests the modulatory effects of ppGpp in gene expression regulated by CRP and H-NS. The method described here may be applicable to many other proteins.

PnBA-b-PNIPAM-b-PDMAEA Thermo-Responsive Triblock Terpolymers and Their Quaternized Analogs as Gene and Drug Delivery Vectors

In this work, the ability of thermo-responsive poly [butyl acrylate-b-N-isopropylacrylamide-b-2-(dimethylamino) ethyl acrylate] (PnBA-b-PNIPAM-b-PDMAEA) triblock terpolymer self-assemblies, as well as of their quaternized analogs (PnBA-b-PNIPAM-b-QPDMAEA), to form polyplexes with DNA through electrostatic interactions was examined. Terpolymer/DNA polyplexes were prepared in three different amine over phosphate group ratios (N/P), and linear DNA with a 2000 base pair length was used. In aqueous solutions, the terpolymers formed aggregates of micelles with mixed PNIPAM/(Q)PDMAEA coronas and PnBA cores. The PnBA-b-PNIPAM-b-PDMAEA terpolymers’ micellar aggregates were also examined as carriers for the model hydrophobic drug curcumin (CUR). The complexation ability of the terpolymer with DNA was studied by UV–Vis spectroscopy and fluorescence spectroscopy by investigating ethidium bromide quenching. Fluorescence was also used for the determination of the intrinsic fluorescence of the CUR-loaded micellar aggregates. The structural characteristics of the polyplexes and the CUR-loaded aggregates were investigated by dynamic and electrophoretic light scattering techniques. Polyplexes were found to structurally respond to changes in solution temperature and ionic strength, while the intrinsic fluorescence of encapsulated CUR was increased at temperatures above ambient.