[32] Magnetic bead assay for characterization of microtubule-membrane interactions

Abstract The characterization of microbial community dynamics using genomic methods is rapidly expanding, impacting many fields including medical, ecological, and environmental research and applications. One of the biggest challenges for such studies is the isolation of environmental DNA (eDNA) from a variety of samples, diverse microbes, and widely variable community compositions. The current study developed environmentally friendly, user safe, economical, and high throughput eDNA extraction methods for mixed aquatic microbial communities and tested them using 16 s rRNA gene meta-barcoding. Five different lysis buffers including (1) cetyltrimethylammonium bromide (CTAB), (2) digestion buffer (DB), (3) guanidinium isothiocyanate (GITC), (4) sucrose lysis (SL), and (5) SL-CTAB, coupled with four different purification methods: (1) phenol-chloroform-isoamyl alcohol (PCI), (2) magnetic Bead-Robotic, (3) magnetic Bead-Manual, and (4) membrane-filtration were tested for their efficacy in extracting eDNA from recreational freshwater samples. Results indicated that the CTAB-PCI and SL-Bead-Robotic methods yielded the highest genomic eDNA concentrations and succeeded in detecting the core microbial community including the rare microbes. However, our study recommends the SL-Bead-Robotic eDNA extraction protocol because this method is safe, environmentally friendly, rapid, high-throughput and inexpensive.

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Development and characterization of a magnetic bead-quantum dot nanoparticles based assay capable of Escherichia coli O157:H7 quantification

Analytica Chimica Acta ◽

10.1016/j.aca.2010.07.046 ◽

2010 ◽

Vol 677 (1) ◽

pp. 90-96 ◽

Cited By ~ 39

Author(s):

Gha-Young Kim ◽

Ahjeong Son

Keyword(s):

Escherichia Coli ◽

Quantum Dot ◽

Magnetic Bead ◽

Escherichia Coli O157

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Structural Basis for Env Incorporation into HIV-1 Particles

Proceedings ◽

10.3390/proceedings2020050114 ◽

2020 ◽

Vol 50 (1) ◽

pp. 114

Author(s):

R. Elliot Murphy ◽

Alexandra B. Samal ◽

Gunnar Eastep ◽

Ruba H. Ghanam ◽

Peter E. Prevelige ◽

...

Keyword(s):

Structural Characterization ◽

Late Phase ◽

Three Dimensional ◽

Dimensional Structure ◽

Structural Basis ◽

Membrane Interactions ◽

Env Protein ◽

Gag Assembly ◽

Hiv 1

During the late phase of the HIV-1 replication cycle, the Gag polyproteins are transported to the plasma membrane (PM) for assembly. Gag targeting and assembly on the PM is dependent on interactions between its matrix (MA) domain and phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). Subsequent to Gag assembly, the envelope (Env) protein is recruited to the PM for incorporation into virus particles. Evidence suggests that the incorporation of the Env protein is mediated by interactions between the MA domain of Gag and the cytoplasmic tail of the gp41 subunit of Env (gp41CT), a mechanism that remains to be elucidated. Trimerization of the MA domain of Gag appears to be an obligatory step for this interaction. The interplay between gp41CT, the MA trimer, and the membrane has yet to be determined. Our lab has pioneered methods and approaches to investigate, at the molecular level, how the retroviral MA domains of Gag interact with membranes, a key requirement for understanding the Gag assembly and Env incorporation. Herein, we devised innovative approaches that will enable the structural characterization of the gp41CT–MA–membrane interactions. We employed structural biology (NMR and cryo-electron microscopy, biophysical methods, and biochemical tools to generate a macromolecular picture of how the MA domain of Gag binds to the membrane and how it interacts with gp41CT. To this end, we: (i) determined the three-dimensional structure of HIV-1 gp41CT and characterized its interaction with the membrane, (ii) engineered trimeric constructs of gp41CT and the MA to recapitulate the native and functional states of the proteins, and (iii) utilized membrane nanodisc technology to anchor the MA and gp41CT proteins. Our studies will allow for a detailed structural characterization of the gp41CT–MA–membrane interactions, which will advance our knowledge of HIV-1 Gag assembly and Env incorporation.

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