Development of ion torrent-based targeted next-generation sequencing panel for identification of animal species in pet foods.

Manufacturers may intentionally or unintentionally incorporate ingredients not specified on the label of canned pet foods. Including any unacknowledged ingredients in a food product is considered food fraud or misbranding. Contamination of pet foods may occur in the processing of the foods, including potential cross-contamination in packaging facilities. Of the methods available to identify meat species in food products, Sanger sequencing and several next-generation sequencing methods are available, but there are limitations including the number of targets analyzed at a time and the method specificity. In this study, we developed a targeted next-generation sequencing panel to detect meat species in canned pet foods using Ion Torrent technology. The panel contains multiple primers targeting mitochondrial genes from as many as 27 animal species, of which 7 major animal species were validated. The meat species targets could be identified from samples spiked with as low as 0.01% w/w of the contaminating meat species in a vegetarian food matrix material. Targeted NGS in the current study enriches species-specific multiple target areas in the mitochondrial genome of the target material, which gives high accuracy in the sequencing results.

The role of mecA and blaZ regulatory elements in mecA expression by regional clones of methicillin-resistant Staphylococcus pseudintermedius.

Two major regional clones of methicillin-resistant Staphylococcus pseudintermedius (MRSP) have been identified in Europe and North America. They are designated multilocus sequence types (ST) 71 and 68 and contain staphylococcal chromosome cassette (SCCmec) types II-III and V(T), respectively. One notable difference between the two clones is a deletion in the mecI/mecR1 regulatory apparatus of ST 68 SCCmec V(T). This deletion in analogous methicillin-resistant Staphylococcus aureus (MRSA) results in more responsive and greater expression of the mecA encoded penicillin-binding protein 2a, and is associated with SCCmec types occurring in community-acquired MRSA lineages. The aim of this study was to characterize mec and bla regulatory apparatuses in MRSP and determine their effects on expression of mecA. Seventeen S. pseudintermedius isolates representing nine methicillin-resistant ST lineages were screened for the presence of the repressors blaI and mecI and sensors blaR1 and mecR1. The bla and mec operons for each isolate were sequenced and compared for homology between the repressor open-reading frames (ORF), sensor ORFs, and mecA promoter regions. A real-time reverse transcriptase PCR expression assay was developed, validated and applied to nine isolates determining the effect of oxacillin induction on mecA transcription. Significant differences were found in mecA expression between isolates with a full regulatory complement (mecI/mecR1 and blaI/blaR1) and those with truncated and/or absent regulatory elements. Isolates representative of European and North American MRSP ST regional clones have dissimilar mecA responses to oxacillin.

The Intrinsic Incompressibility of Osteoblast-like Cells.

This paper presents a new methodology, apparatus design, and the experimental results of ongoing research into the measurement of the mechanical properties of musculoskeletal tissue at the cellular level. A microchamber was constructed that provides a controlled hydrostatic pressure environment for these cells where optical sectioning, via epifluorescence microscopy, was used to acquire volume information about the individual cell. The microchamber was integrated into a hydraulic system that, via computer control, provided a regulated adjustable hydrostatic pressure environment for living cells suspended in culture media. The techniques applied in this study include fluorescent labeling of the cell volume, hydrostatic pressure application, optical sectioning, and digital volume reconstruction. To determine the mechanical response (compressibility) of cultured MG-63 osteoblast-like cells under physiologically high hydrostatic pressures two experiments were devised: In the first experiment changes in volume of 10 cells were measured as the applied hydrostatic pressure was increased from 0 to 7 MPa. Volume changes in response to pressure magnitudes were not significant (p > 0.49). In the second experiment, the mechanical role of the plasma membrane to act as a supportive component in cell compressibility was studied by permeabilizing the membrane of six cells and again applying hydrostatic pressure. Again, no significant volume differences between pressurized and unpressurized cells were found (p > 0.46). A retrospective power analysis of the results of the first and second experiments indicates that the sample size was sufficient. The results of this study show that MG-63 osteoblast-like cells are intrinsically incompressible in the 0-7 MPa hydrostatic pressure range. They also support the hypothesis that the plasma membrane plays an insignificant mechanical role in terms of cell compressibility.