Reviewer
Yan Wang
  • Post-Doc, Stanford University
Research fields
  • Neuroscience
A Guideline for Assessment and Characterization of Bacterial Biofilm Formation in the Presence of Inhibitory Compounds
Authors:  Bassam A. Elgamoudi and Victoria Korolik, date: 11/05/2023, view: 861, Q&A: 0

Campylobacter jejuni, a zoonotic foodborne pathogen, is the worldwide leading cause of acute human bacterial gastroenteritis. Biofilms are a significant reservoir for survival and transmission of this pathogen, contributing to its overall antimicrobial resistance. Natural compounds such as essential oils, phytochemicals, polyphenolic extracts, and D-amino acids have been shown to have the potential to control biofilms formed by bacteria, including Campylobacter spp. This work presents a proposed guideline for assessing and characterizing bacterial biofilm formation in the presence of naturally occurring inhibitory molecules using C. jejuni as a model. The following protocols describe: i) biofilm formation inhibition assay, designed to assess the ability of naturally occurring molecules to inhibit the formation of biofilms; ii) biofilm dispersal assay, to assess the ability of naturally occurring inhibitory molecules to eradicate established biofilms; iii) confocal laser scanning microscopy (CLSM), to evaluate bacterial viability in biofilms after treatment with naturally occurring inhibitory molecules and to study the structured appearance (or architecture) of biofilm before and after treatment.

Triplet-primed PCR and Melting Curve Analysis for Rapid Molecular Screening of Spinocerebellar Ataxia Types 1, 2, and 3
Authors:  Mulias Lian, Mingjue Zhao, Gui-Ping Phang, Indhu-Shree Rajan-Babu and Samuel S. Chong, date: 06/20/2023, view: 436, Q&A: 0

There are more than 40 types of spinocerebellar ataxia (SCA), most of which are caused by abnormal expansion of short tandem repeats at various gene loci. These phenotypically similar disorders require molecular testing at multiple loci by fluorescent PCR and capillary electrophoresis to identify the causative repeat expansion. We describe a simple strategy to screen for the more common SCA1, SCA2, and SCA3 by rapidly detecting the abnormal CAG repeat expansion at the ATXN1, ATXN2, and ATXN3 loci using melting curve analysis of triplet-primed PCR products. Each of the three separate assays employs a plasmid DNA carrying a known repeat size to generate a threshold melt peak temperature, which effectively distinguishes expansion-positive samples from those without a repeat expansion. Samples that are screened positive based on their melt peak profiles are subjected to capillary electrophoresis for repeat sizing and genotype confirmation. These screening assays are robust and provide accurate detection of the repeat expansion while eliminating the need for fluorescent PCR and capillary electrophoresis for every sample.

A Rapid FRET Real-Time PCR Protocol for Simultaneous Quantitative Detection and Discrimination of Human Plasmodium Parasites

Malaria is the most important parasitic disease worldwide, and accurate diagnosis and treatment without delay are essential for reducing morbidity and mortality, especially in P. falciparum malaria. Real-time PCR is highly sensitive and highly specific, therefore an excellent diagnostic tool for laboratory detection and species-specific diagnosis of malaria, especially in non-endemic regions where experience in microscopic malaria diagnostics is limited. In contrast to many other real-time PCR protocols, our new fluorescence resonance energy transfer-based real-time PCR (FRET-qPCR) allows the quantitative and species-specific detection of all human Plasmodium spp. in one run. Species identification is based on single nucleotide polymorphisms (SNPs) within the MalFL probe, detectable by melting curve analysis. A significant advantage of our FRET-qPCR is the short turn-around time of less than two hours, including DNA extraction, which qualifies it for routine diagnostics. Rapid and reliable species-specific malaria diagnosis is important, because treatment is species-dependent. Apart from first-line diagnosis, the quantitative results of our new FRET-qPCR can be helpful in therapy control, to detect early treatment failure.


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Microtubule Seeded-assembly in the Presence of Poorly Nucleating Nucleotide Analogues
Authors:  Siou Ku, Claire Heichette, Laurence Duchesne and Denis Chrétien, date: 08/20/2020, view: 2798, Q&A: 0
Microtubule dynamic instability is driven by the hydrolysis of the GTP bound to the β-subunit of the α-β tubulin heterodimer. Nucleotide analogues are commonly used to mimic the different steps of the tubulin GTPase cycle, but most of them are poor microtubule nucleators. Usually, microtubule assembly is seeded by guanylyl-(α, β)-methylene-diphosphonate (GMPCPP) or glycerol that can be limiting factors in monitoring the effect of other nucleotide analogs on their polymerization. Here, we describe a protocol that allows the assembly of microtubules in the presence of nucleotide analogues without the need of heterogeneous seeds and at a low final glycerol concentration. Microtubules are first assembled in the presence of the analogue of interest and glycerol to promote assembly. These microtubules are then sonicated to produce seeds that will be used to assemble microtubules in the absence of glycerol. This strategy produces homogeneous nucleotide-bound microtubules that can be further analyzed by biochemical or structural methods such as cryo-electron microscopy.
F-actin Bundle Sedimentation Assay
Authors:  Shan-Shan Lin, Mei-Chun Chuang and Ya-Wen Liu, date: 11/05/2019, view: 4675, Q&A: 0
Understanding the molecular mechanism governing the higher-order regulation of actin dynamics requires chemically-defined and quantitative assays. Recently, the membrane remodeling large GTPase, dynamin, has been identified as a new actin cross-linking molecule. Dynamin regulates actin cytoskeleton through binding to, self-assembling around, and aligning them into actin bundles. Here we utilize dynamin as an example and present a simple protocol to analyze the actin bundling activity in vitro. This protocol details the method for F-actin reconstitution as well as quantitative and qualitative analyses for actin bundling activity of dynamins. Measurement of the actin bundling activity of other actin-binding proteins may also be applied to this protocol with appropriate adjustments depending on the protein of interest.
Determination of Survival of Wildtype and Mutant Escherichia coli in Soil
Authors:  Yinka Somorin and Conor O'Byrne, date: 07/20/2017, view: 7783, Q&A: 0
E. coli resides in the gastrointestinal tract of humans and other warm-blooded animals but recent studies have shown that E. coli can persist and grow in various external environments including soil. The general stress response regulator, RpoS, helps E. coli overcome various stresses, however its role in soil survival was unknown. This soil survival assay protocol was developed and used to determine the role of the general stress response regulator, RpoS, in the survival of E. coli in soil. Using this soil survival assay, we demonstrated that RpoS was important for the survival of E. coli in soil. This protocol describes the development of the soil survival assay especially the recovery of E. coli inoculated into soil and can be adapted to allow further investigations into the survival of other bacteria in soil.
Expression and Purification of Cyanobacterial Circadian Clock Protein KaiC and Determination of Its Auto-phosphatase Activity
Authors:  Qiang Chen, Lingling Yu, Xiao Tan and Sen Liu, date: 02/20/2017, view: 7388, Q&A: 0
Circadian rhythms are biological processes displaying an endogenous oscillation with a period of ~24 h. They allow organisms to anticipate and get prepared for the environmental changes caused mainly by the rotation of Earth. Circadian rhythms are driven by circadian clocks that consist of proteins, DNA, and/or RNA. Circadian clocks of cyanobacteria are the simplest and one of the best studied models. They contain the three clock proteins KaiA, KaiB, and KaiC which can be used for in vitro reconstitution experiments and determination of the auto-phosphatase activity of KaiC as described in this protocol.
Analytical Gel Filtration for Probing Heavy Metal Transfer between Proteins
Authors:  Steffen Lorenz Drees and Mathias Lübben, date: 08/05/2016, view: 9907, Q&A: 0
Heavy metals can cause damage to biomolecules such as proteins and DNA in multiple ways. Cells therefore strive for keeping intracellular (heavy) metal ions bound to specific proteins that are capable of handling detoxification, export or integration as cofactors. Metal binding proteins usually provide specific coordination sites that bind certain ions with ultrahigh affinity, with the thermodynamic driving force being the stability of organometallic complexes. However, the metal binding properties of these proteins can be highly variable. Therefore the transfer of specific ions between separate proteins or even between distinct binding sites located on one and the same protein does not always follow affinity gradients, but depends on particular protein interactions that are difficult to predict. We established a method suitable to probe metal transfer between two proteins, provided the proteins are amenable to purification and in vitro handling. It consists of the loading with metals, the co-incubation and the separation of metal-exchanging proteins with subsequent determination of bound metal content. The method is exemplified by experimental data of ours probing the transfer of copper(I) between the membrane-extrinsic metal binding domain MBD2 and the transmembrane domain of CopA, a copper export ATPase from Escherichia coli (Drees et al., 2015).
Quantification of Respiratory Activity in Biofilms
Authors:  Cláudia N. H. Marques and Scott A. Craver, date: 09/20/2015, view: 10004, Q&A: 1
Bacteria live mostly as biofilms, not as planktonic cell populations. Bacterial cells living as biofilms are known to be in different physiological status. Persister cells are one of such physiological conditions and they are recognized as to be a stochastically produced sub-population of non-growing bacterial cells. The following protocol describes a method to determine the respiratory activity of cells within biofilms.
Isolation of Persister Cells from Biofilm and Planktonic Populations of Pseudomonas aeruginosa
Author:  Cláudia N. H. Marques, date: 09/20/2015, view: 12100, Q&A: 0
Persister cells are a stochastically produced sub-population of non-growing bacterial cells. Recently these cells have been more widely studied due to the recognition that they are tolerant to antimicrobials and thus, play a major role in the resilience of bacterial populations to antimicrobials, particularly in chronic biofilm infections. The following protocol describes the isolation/selection of persister cell sub-populations of Pseudomonas aeruginosa present in biofilms (sessile) and planktonic populations (free-living).
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