Biomedical subjects

Molly A Hughes

Publications and source records attributed to Molly A Hughes.

3 recordsLinked to original sources

PubMed2006

A fully integrated microfluidic genetic analysis system with sample-in-answer-out capability.

We describe a microfluidic genetic analysis system that represents a previously undescribed integrated microfluidic device capable of accepting whole blood as a crude biological sample with the endpoint generation of a genetic profile. Upon loading the sample, the glass microfluidic genetic analysis system device carries out on-chip DNA purification and PCR-based amplification, followed by separation and detection in a manner that allows for microliter samples to be screened for infectious pathogens with sample-in-answer-out results in < 30 min. A single syringe pump delivers sample/reagents to the chip for nucleic acid purification from a biological sample. Elastomeric membrane valving isolates each distinct functional region of the device and, together with resistive flow, directs purified DNA and PCR reagents from the extraction domain into a 550-nl chamber for rapid target sequence PCR amplification. Repeated pressure-based injections of nanoliter aliquots of amplicon (along with the DNA sizing standard) allow electrophoretic separation and detection to provide DNA fragment size information. The presence of Bacillus anthracis (anthrax) in 750 nl of whole blood from living asymptomatic infected mice and of Bordetella pertussis in 1 microl of nasal aspirate from a patient suspected of having whooping cough are confirmed by the resultant genetic profile.

DNA↗

PubMed2005

MyD88-dependent signaling contributes to protection following Bacillus anthracis spore challenge of mice: implications for Toll-like receptor signaling.

Bacillus anthracis is a spore-forming, gram-positive organism that is the causative agent of the disease anthrax. Recognition of Bacillus anthracis by the host innate immune system likely plays a key protective role following infection. In the present study, we examined the role of TLR2, TLR4, and MyD88 in the response to B. anthracis. Heat-killed Bacillus anthracis stimulated TLR2, but not TLR4, signaling in HEK293 cells and stimulated tumor necrosis factor alpha (TNF-alpha) production in C3H/HeN, C3H/HeJ, and C57BL/6J bone marrow-derived macrophages. The ability of heat-killed B. anthracis to induce a TNF-alpha response was preserved in TLR2-/- but not in MyD88-/- macrophages. In vivo studies revealed that TLR2-/- mice and TLR4-deficient mice were resistant to challenge with aerosolized Sterne strain spores but MyD88-/- mice were as susceptible as A/J mice. We conclude that, although recognition of B. anthracis occurs via TLR2, additional MyD88-dependent pathways contribute to the host innate immune response to anthrax infection.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Identification of Entamoeba histolytica thiol-specific antioxidant as a GalNAc lectin-associated protein.

Entamoeba histolytica is a human intestinal parasite that causes amebic dysentery. A cell surface amebic adhesin, the galactose and N-acetyl-D-galactosamine inhibitable (GalNAc) lectin mediates amebic adherence to and contact-dependent killing of host cells. Previous work has suggested that the GalNAc lectin transduces signals via protein interactions with its short cytoplasmic domain. We used a yeast two-hybrid system to screen an E. histolytica cDNA library for proteins that interact with the GalNAc lectin cytoplasmic domain. One isolate was the E. histolytica thiol-specific antioxidant (TSA). TSA is an enzyme that detoxifies hydrogen peroxide. TSA did not interact in yeast two-hybrid experiments with a mutant version of the lectin cytoplasmic domain, confirming the specificity of the lectin-TSA interaction. Furthermore, mutational analyses of the TSA isolate demonstrated that an in-frame five amino acid sequence introduced between amino acids 61-62 yielded a TSA mutant that did not interact with the lectin cytoplasmic domain upon expression in the yeast two-hybrid system. The association of TSA and GalNAc lectin was further supported by co-immunoaffinity purification. Confocal microscopy demonstrated co-localization of TSA and GalNAc lectin at sites of ameba:host cell contact. Recruitment of TSA by the GalNAc lectin suggests a novel mechanism of parasite defense against reactive oxygen intermediates generated by host peripheral mononuclear cells.

Acetylgalactosamine↗