Selection of suitable reference genes for quantitative gene expression studies in milk somatic cells of lactating cows (Bos indicus).

We assessed the suitability of 9 internal control genes (ICG) in milk somatic cells of lactating cows to find suitable reference genes for use in quantitative PCR (qPCR). Eighteen multiparous lactating Sahiwal cows were used, 6 in each of 3 lactation stages: early (25 ± 5 d in milk), mid (160 ± 15 d in milk), and late (275 ± 25 d in milk) lactation. Nine candidate reference genes [glyceraldehyde 3-phosphate dehydrogenase (GAPDH), protein phosphatase 1 regulatory subunit 11 (PPP1R11), ß-actin (ACTB), ß-2 microglobulin (B2M), 40S ribosomal protein S15a (RPS15A), ubiquitously expressed transcript (UXT), mitochondrial GTPase 1 (MTG1), 18S rRNA (RN18S1), and ubiquitin (UBC)] were evaluated. Three genes, ß-casein (CSN2), lactoferrin (LTF), and cathelicidin (CAMP) were chosen as target genes. Very high amplification was observed in 7 ICG and very low level amplification was observed in 2 ICG (UXT and MTG1). Thus, UXT and MTG1 were excluded from further analysis. The qPCR data were analyzed by 2 software packages, geNorm and NormFinder, to determine suitable reference genes, based on their stability and expression. Overall, PPP1R11, ACTB, UBC, and GAPDH were stably expressed among all candidate reference genes. Therefore, these genes could be used as ICG for normalization of qPCR data in milk somatic cells through lactation.

Use of microwells to investigate the effect of quorum sensing on growth and antigen production in Bacillus anthracis Sterne 34F2.

AIM: The aim of this study was to investigate the role of quorum sensing in Bacillus anthracis growth and toxin production. METHODS AND RESULTS: A microwell plate culture method was developed to simulate the normal UK-licensed anthrax vaccine production run. Once established, sterile supernatant additions from a previous B. anthracis culture were made, and reductions in lag phase and early stimulation of the anthrax toxin component protective antigen (PA) were monitored using ELISA. The addition of the quorum-sensing inhibitor, fur-1, prolonged the lag phase and impeded PA production. Spin filters of various sizes were used to identify the molecular weight fraction of the sterile supernatant responsible for the autoinducer effect. A weight fraction between 5 and 10 kDa was responsible for the autoinducer effect; however, further identification using mass spectroscopy proved inconclusive. CONCLUSIONS: Quorum sensing mediated by the autoinducer two molecule plays a significant role in both B. anthracis growth and toxin production. SIGNIFICANCE AND IMPACT OF THE STUDY: While genomic analysis has eluded to the importance of LuxS and quorum sensing in anthrax, this is the first analysis using a production strain of B. anthracis and a quorum-sensing inhibitor to monitor the effect on growth and toxin production. This gives insights into anthrax pathogenicity and vaccine manufacture.