Applied Biosystems

About: Applied Biosystems is a based out in . It is known for research contribution in the topics: Mass spectrometry & Nucleic acid. The organization has 1521 authors who have published 1579 publications receiving 285423 citations.

High-throughput detection of West Nile virus RNA

Abstract: The recent outbreaks of West Nile virus (WNV) in the northeastern United States and other regions of the world have made it essential to develop an efficient protocol for surveillance of WNV. In the present report, we describe a high-throughput procedure that combines automated RNA extraction, amplification, and detection of WNV RNA. The procedure analyzed 96 samples in approximately 4.5 h. A robotic system, the ABI Prism 6700 Automated Nucleic Acid workstation, extracted RNA and set up reactions for real-time reverse transcription (RT)-PCR in a 96-well format. The robot extracted RNA with a recovery as efficient as that of a commercial RNA extraction kit. A real-time RT-PCR assay was used to detect and quantitate WNV RNA. Using in vitro transcribed RNA, we estimated the detection limit of the real-time RT-PCR to be approximately 40 copies of RNA. A standard RT-PCR assay was optimized to a sensitivity similar to that of the real-time RT-PCR. The standard assay can be reliably used to test a small number of samples or to confirm previous test results. Using internal primers in a nested RT-PCR, we increased the sensitivity by approximately 10-fold compared to that of the standard RT-PCR. The results of the study demonstrated for the first time that the use of an automated system for the purpose of large-scale viral RNA surveillance dramatically increased the speed and efficiency of sample throughput for diagnosis.

Loss of functional ELOVL4 depletes very long-chain fatty acids (≥C28) and the unique ω-O-acylceramides in skin leading to neonatal death

Abstract: Mutations in elongation of very long-chain fatty acid-4 (ELOVL4) are associated with autosomal dominant Stargardt-like macular degeneration (STGD3), with a five base-pair (5 bp) deletion mutation resulting in the loss of 51 carboxy-terminal amino acids and truncation of the protein. In addition to the retina, Elovl4 is expressed in a limited number of mammalian tissues, including skin, with unknown function(s). We generated a knock-in mouse model with the 5-bp deletion in the Elovl4 gene. As anticipated, mice carrying this mutation in the heterozygous state (Elovl4(+/del)) exhibit progressive photoreceptor degeneration. Unexpectedly, homozygous mice (Elovl4(del/del)) display scaly, wrinkled skin, have severely compromised epidermal permeability barrier function, and die within a few hours after birth. Histopathological evaluation of the Elovl4(del/del) pups revealed no apparent abnormality(ies) in vital internal organs. However, skin histology showed an abnormally-compacted outer epidermis [stratum corneum (SC)], while electron microscopy revealed deficient epidermal lamellar body contents, and lack of normal SC lamellar membranes that are essential for permeability barrier function. Lipid analyses of epidermis from Elovl4(del/del) mice revealed a global decrease in very long-chain fatty acids (VLFAs) (i.e., carbon chain > or =C28) in both the ceramide/glucosylceramide and the free fatty-acid fractions. Strikingly, Elovl4(del/del) skin was devoid of the epidermal-unique omega-O-acylceramides, that are key hydrophobic components of the extracellular lamellar membranes in mammalian SC. These findings demonstrate that ELOVL4 is required for generating VLFA critical for epidermal barrier function, and that the lack of epidermal omega-O-acylceramides is incompatible with survival in a desiccating environment.

C-terminal signal sequence promotes virulence factor secretion in Mycobacterium tuberculosis.

Abstract: Mycobacterium tuberculosis uses the ESX-1/Snm system [early secreted antigen 6 kilodaltons (ESAT-6) system 1/secretion in mycobacteria] to deliver virulence factors into host macrophages during infection. Despite its essential role in virulence, the mechanism of ESX-1 secretion is unclear. We found that the unstructured C terminus of the CFP-10 substrate was recognized by Rv3871, a cytosolic component of the ESX-1 system that itself interacts with the membrane protein Rv3870. Point mutations in the signal that abolished binding of CFP-10 to Rv3871 prevented secretion of the CFP-10 (culture filtrate protein, 10 kilodaltons)/ESAT-6 virulence factor complex. Attachment of the signal to yeast ubiquitin was sufficient for secretion from M. tuberculosis cells, demonstrating that this ESX-1 signal is portable.