Transcriptome analysis of a respiratory Saccharomyces cerevisiae strain suggests the expression of its phenotype is glucose insensitive and predominantly controlled by Hap4, Cat8 and Mig1

BACKGROUND: We previously described the first respiratory Saccharomyces cerevisiae strain, KOY.TM6*P, by integrating the gene encoding a chimeric hexose transporter, Tm6*, into the genome of an hxt null yeast. Subsequently we transferred this respiratory phenotype in the presence of up to 50 g/L glucose to a yeast strain, V5 hxt1-7Delta, in which only HXT1-7 had been deleted. In this study, we compared the transcriptome of the resultant strain, V5.TM6*P, with that of its wild-type parent, V5, at different glucose concentrations. RESULTS: cDNA array analyses revealed that alterations in gene expression that occur when transitioning from a respiro-fermentative (V5) to a respiratory (V5.TM6*P) strain, are very similar to those in cells undergoing a diauxic shift. We also undertook an analysis of transcription factor binding sites in our dataset by examining previously-published biological data for Hap4 (in complex with Hap2, 3, 5), Cat8 and Mig1, and used this in combination with verified binding consensus sequences to identify genes likely to be regulated by one or more of these. Of the induced genes in our dataset, 77% had binding sites for the Hap complex, with 72% having at least two. In addition, 13% were found to have a binding site for Cat8 and 21% had a binding site for Mig1. Unexpectedly, both the up- and down-regulation of many of the genes in our dataset had a clear glucose dependence in the parent V5 strain that was not present in V5.TM6*P. This indicates that the relief of glucose repression is already operable at much higher glucose concentrations than is widely accepted and suggests that glucose sensing might occur inside the cell. CONCLUSION: Our dataset gives a remarkably complete view of the involvement of genes in the TCA cycle, glyoxylate cycle and respiratory chain in the expression of the phenotype of V5.TM6*P. Furthermore, 88% of the transcriptional response of the induced genes in our dataset can be related to the potential activities of just three proteins: Hap4, Cat8 and Mig1. Overall, our data support genetic remodelling in V5.TM6*P consistent with a respiratory metabolism which is insensitive to external glucose concentrations.

Tear analysis and lens-tear interactions:part II. Ocular lipids-nature and fate of meibomian gland phospholipids

Purpose: Published data indicate that the polar lipid content of human meibomian gland secretions (MGS) could be anything between 0.5% and 13% of the total lipid. The tear film phospholipid composition has not been studied in great detail and it has been understood that the relative proportions of lipids in MGS would be maintained in the tear film. The purpose of this work was to determine the concentration of phospholipids in the human tear film. Methods: Liquid chromatography mass spectrometry (LCMS) and thin layer chromatography (TLC) were used to determine the concentration of phospholipid in the tear film. Additionally, an Amplex Red phosphatidylcholine-specific phospholipase C (PLC) assay kit was used for determination of the activity of PLC in the tear film. Results: Phospholipids were not detected in any of the tested human tear samples with the low limit of detection being 1.3 µg/mL for TLC and 4 µg/mL for liquid chromatography mass spectrometry. TLC indicated that diacylglycerol (DAG) may be present in the tear film. PLC was in the tear film with an activity determined at approximately 15 mU/mL, equivalent to the removal of head groups from phosphatidylcholine at a rate of approximately 15 µM/min. Conclusions: This work shows that phospholipid was not detected in any of the tested human tear samples (above the lower limits of detection as described) and suggests the presence of DAG in the tear film. DAG is known to be at low concentrations in MGS. These observations indicate that PLC may play a role in modulating the tear film phospholipid concentration.

Randomised masked clinical trial of the MGDRx eyebag for the treatment of meibomian gland dysfunction-related evaporative dry eye

Background/aims To investigate the efficacy and safety of the MGDRx EyeBag (The Eyebag Company, Halifax, UK) eyelid warming device. Methods Twenty-five patients with confirmed meibomian gland dysfunction (MGD)-related evaporative dry eye were enrolled into a randomised, single masked, contralateral clinical trial. Test eyes received a heated device; control eyes a non-heated device for 5 min twice a day for 2 weeks. Efficacy (ocular symptomology, noninvasive break-up time, lipid layer thickness, osmolarity, meibomian gland dropout and function) and safety (visual acuity, corneal topography, conjunctival hyperaemia and staining) measurements were taken at baseline and follow-up. Subsequent patient device usage and ocular comfort was ascertained at 6 months. Results Differences between test and control eyes at baseline were not statistically signi ficant for all measurements ( p>0.05). After 2 weeks, statistically significant improvements occurred in all efficacy measurements in test eyes ( p<0.05). Visual acuity and corneal topography were unaffected (p>0.05). All patients maintained higher ocular comfort after 6 months ( p<0.05), although the bene fit was greater in those who continued usage 1-8 times a month (p<0.001). Conclusions The MGDRx EyeBag is a safe and effective device for the treatment of MGD-related evaporative dry eye. Subjective benefit lasts at least 6 months, aided by occasional retreatment. Trial registration number NCT01870180.

Microwave decontamination of eyelid warming devices for the treatment of meibomian gland dysfunction

PURPOSE: The role of bacteria in meibomian gland dysfunction is unclear, yet contamination of compresses used as treatment may exacerbate this condition. This study therefore determined the effect of heating on bacteria on two forms of compress. METHODS: Cotton flannels and MGDRx EyeBags (eyebags) were inoculated by adding experimental inoculum (Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa; one species for each set of 3 eyebags and flannels). One of each were then randomised in to 3 groups: no heating (control); therapeutic (47.4±0.7°C); or sanitisation (68±1.1°C). After treatment, bacteria cell numbers were calculated. The experiment was repeated in triplicate. RESULTS: There was a statistically significant difference between each treatment with the eyebag for S. aureus (control=7.15±0.11logC/ml, therapeutic heating=5.24±0.59logC/ml, sanitisation heating=3.48±1.43logC/ml; P<0.001) and S. pyogenes (7.36±0.13, 5.73±0.26, 4.75±0.54; P<0.001). P. aeruginosa also showed a significant reduction (P<0.001) from control (6.39±0.34) to therapeutic (0.33±0.26) and sanitisation (0.33±0.21), but the latter were similar (P=1.000). For the flannels, there was significant difference between each treatment for S. aureus (6.89±0.46, 3.96±1.76, 0.42±0.90; P<0.001). For S. pyogenes, there was a significant reduction (P<0.001) from control (7.51±0.10) to therapeutic (5.91±0.62) and sanitisation (5.18±0.8), but the latter were similar (P=0.07). For P. aeruginosa, there was a significant difference (P<0.001) from control (7.15±0.36) to sanitisation (5.83±0.44); but not to therapeutic (6.84±0.31) temperatures (P=0.07). CONCLUSIONS: Therapeutic heating produces a significant reduction in bacteria on the eyebags, but only sanitisation heating appears effective for flannels. However, patients should be advised to heat the eyebag to sanitisation temperatures on initial use.