A New, Quick, and Simple Protocol to Evaluate Microalgae Polysaccharide Composition.

In this work, a new methodological approach, relying on the high specificity of enzymes in a complex mixture, was developed to estimate the composition of bioactive polysaccharides produced by microalgae, directly in algal cultures. The objective was to set up a protocol to target oligomers commonly known to be associated with exopolysaccharides' (EPS) nutraceutical and pharmaceutical activities (i.e., rhamnose, fucose, acidic sugars, etc.) without the constraints classically associated with chromatographic methods, while maintaining a resolution sufficiently high to enable their monitoring in the culture system. Determination of the monosaccharide content required the application of acid hydrolysis (2 M trifluoroacetic acid) followed by NaOH (2 M) neutralization. Quantification was then carried out directly on the fresh hydrolysate using enzyme kits corresponding to the main monosaccharides in a pre-determined composition of the polysaccharides under analysis. Initial results showed that the enzymes were not sensitive to the presence of TFA and NaOH, so the methodology could be carried out on fresh hydrolysate. The limits of quantification of the method were estimated as being in the order of the log of nanograms of monosaccharides per well, thus positioning it among the chromatographic methods in terms of analytical performance. A comparative analysis of the results obtained by the enzymatic method with a reference method (high-performance anion-exchange chromatography) confirmed good recovery rates, thus validating the closeness of the protocol. Finally, analyses of raw culture media were carried out and compared to the results obtained in miliQ water; no differences were observed. The new approach is a quick, functional analysis method allowing routine monitoring of the quality of bioactive polysaccharides in algal cultures grown in photobioreactors.

Introducing a cost-effective method for purification of bioactive flagellin from several flagellated gram-negative bacteria.

The objective of this study was to introduce a simple and cheap method for purification of flagellin. So, flagellin proteins of Salmonella typhimurium (S. typhimurium), Escherichia coli (E. coli), Citrobacter freundii (C. freundii) and Salmonella typhi (S. typhi) were purified by a modified simple method. Bacterial cultures were precipitated by centrifugation. Precipitates were washed twice and flagellin proteins were detached by shaking vigorously (in PBS pH = 2), and then flagellin proteins were precipitated by ammonium sulfate saturation. Evaluation of purification efficiency and concentration were examined by SDS-PAGE and Bradford assay. Polyclonal antibodies were produced against S. typhimurium FliC and cross-reactivity of anti-S. typhimurium was assessed against other flagellins. Bioactivity of flagellins was evaluated by cell proliferation and IL-8 protein expression assay in HEK293 cells, and also, IL-6 and TNF-α genes expression in chicken cells. Results showed a single band for flagellin proteins of all bacteria on %10 SDS-PAGE, which concentration ranged from 150 to 400 µg/mL. All flagellin proteins increased cell proliferation, and IL-8 levels were increased after treatment by flagellins and levels of IL-6 and TNF-α were increased after treatment with S. typhimurium FliC. All flagellin proteins showed cross-reactivity with antibodies. Findings showed that application of our method, not only reduced time and cost, but also, the purified flagellin proteins had acceptable bioactivity.

Sensitive mutant detection by concentrating mutant DNA with allele-specific capture and its application to analysis of contaminated grains in rice.

KEY MESSAGE: We developed a method for detection of mutants in a large number of plants, and found this method to be applicable to detection of a mutant allele at a concentration of 1/1000. Many techniques for SNP analysis have been developed, but most of these techniques are not so sensitive to be used for detection of mutants in a large number of plants. Although some highly sensitive methods of SNP analysis have been reported, they are costly. In the present study, a method for concentrating mutant DNA was examined for sensitive detection of an SNP allele in a bulked DNA sample. PCR products of mutant alleles were captured by biotin-labeled oligonucleotide conjugated with streptavidin-coated magnetic beads. By repeated captures of each strand and combining both strands, mutant alleles with a concentration of 1/1000 in wild-type alleles were detectable by CAPS or dCAPS analysis. Indirect capture of a mutant allele was possible, but efficiency was slightly lower than that of the direct capture. The developed method was applied to detection of contamination of rice grains by grains of a different cultivar. Possible applications of this method are discussed.

An efficient and cost-effective method for directed mutagenesis at multiple dispersed sites-a case study with Omicron Spike DNA.

Site-directed mutagenesis is an invaluable technique that enables the elucidation of the contribution of specific residues to protein structure and function. The simultaneous introduction of mutations at a large number of sites (>10), singly and in multiple combinations, is often necessary to fully understand the functional contributions. We report a simple, efficient, time and cost-effective method to achieve this using commonly available molecular biology reagents and protocols, as an alternative to gene synthesis. We demonstrate this method using the Omicron Spike DNA construct as an example, and create a construct bearing 37 mutations (as compared to wild-type Spike DNA), as well as 4 other constructs bearing subsets of the full spectrum of mutations. We believe that this method can be an excellent alternative to gene synthesis, especially when three or more variants are required.

Direct Detection of Extracellular Vesicle miRNAs Using a Single-Step RT-qPCR Assay.

Extracellular vesicles (EVs) are biological carriers, and EV-associated miRNAs (EV-miRNAs) are considered as a novel biomarker in multiple diseases. Currently, the column-based purification method is used to purify miRNAs from EVs. However, this method of purification is complex, time-consuming, and expensive. Therefore, a simple and cost-effective single-step quantitative reverse transcription-polymerase chain reaction (RT-qPCR) method is required to detect the expression of EV-miRNAs. This chapter describes a protocol for directly analyzing the EV-miRNAs expression from mouse bronchoalveolar lavage fluid (BALF) and serum without going for an RNA isolation and purification step from EVs. It is an efficient method in several terms such as cost-wise, time, low expertise, and accuracy in results. This method may be helpful in diagnostic blood tests used in medical centers or research laboratories.

A Sensitive and Cost-Effective Chemiluminescence ELISA for Measurement of Amyloid-ß 1-42 Peptide in Human Plasma.

BACKGROUND: Amyloid-ß42 (Aß42) is associated with plaque formation in the brain of patients with Alzheimer's disease (AD). Studies have suggested the potential utility of plasma Aß42 levels in the diagnosis, and in longitudinal study of AD pathology. Conventional ELISAs are used to measure Aß42 levels in plasma but are not sensitive enough to quantitate low levels. Although ultrasensitive assays like single molecule array or immunoprecipitation-mass spectrometry have been developed to quantitate plasma Aß42 levels, the high cost of instruments and reagents limit their use. OBJECTIVE: We hypothesized that a sensitive and cost-effective chemiluminescence (CL) immunoassay could be developed to detect low Aß42 levels in human plasma. METHODS: We developed a sandwich ELISA using high affinity rabbit monoclonal antibody specific to Aß42. The sensitivity of the assay was increased using CL substrate to quantitate low levels of Aß42 in plasma. We examined the levels in plasma from 13 AD, 25 Down syndrome (DS), and 50 elderly controls. RESULTS: The measurement range of the assay was 0.25 to 500 pg/ml. The limit of detection was 1 pg/ml. All AD, DS, and 45 of 50 control plasma showed measurable Aß42 levels. CONCLUSION: This assay detects low levels of Aß42 in plasma and does not need any expensive equipment or reagents. It offers a preferred alternative to ultrasensitive assays. Since the antibodies, peptide, and substrate are commercially available, the assay is well suited for academic or diagnostic laboratories, and has a potential for the diagnosis of AD or in clinical trials.

Detection of high-risk human papillomavirus type 16 and 18 using isothermal helicase-dependent amplification.

Persistent infection with human papillomavirus (HPV) induces cervical cancer. Here, we describe a sensitive, specific, and rapid assay for high-risk HPV16 and 18 detection by isothermal helicase-dependent amplification. This method can be used as cost-effective diagnostic method for low-income countries, where highest incidences worldwide of cervical cancer are registered.