Effects of topical treatment of cannabidiol extract in a unique manuka factor 5 manuka honey carrier on second intention wound healing on equine distal limb wounds: a preliminary study.

OBJECTIVE: Evaluate the effect of topical 1% cannabidiol on second intention wound healing in distal limb wounds of horses. DESIGN: Experimental. ANIMALS: Six Standardbred horses. METHODS: A total of five 2.5 cm × 2.5 cm full thickness skin wounds were created on the dorsomedial aspect of the metacarpi of 6 horses. Wounds were contaminated with faeces on the day of wound creation. Each wound was then assigned to a treatment group; compounded 1% cannabidiol in unique manuka factor (UMF) 5 manuka honey, UMF 5 manuka honey, UMF 20 manuka honey or saline. Each treatment was applied topically daily for a total of 42 days. Legs were bandaged and bandages were changed, daily, for 13 days postoperatively. Digital photographs of each wound were taken on day 1 then weekly for 6 weeks. Wound size, daily healing rate and total time to healing were recorded and compared statistically. RESULTS: Irrespective of the treatment, wounds did not retract as expected in the first 7 days after wound creation. There was no difference in wound area, daily healing rate, days to complete healing between treatment groups. CONCLUSIONS: This preliminary study failed to demonstrate any difference in wound healing variables between treatment groups in this model of second intention wound healing. This was unexpected due to the established effects of UMF 20 manuka honey on wound healing using the same model. This may be due to systemic effects of cannabidiol and study design. Further research into the use of cannabidiol in equine wounds is warranted.

Pilot study to quantify the time to clear dexamethasone from plasma and urine of adult horses following a single nebulisation.

OBJECTIVE: To quantify the time to clear dexamethasone from plasma and urine of horses following a single nebulisation. DESIGN: Experimental using six Standardbred mares. METHODS: Dexamethasone sodium phosphate (0.04 mg/kg) diluted in 0.9% sodium chloride was administered as an aerosol using a Flexineb E2® nebuliser. Blood samples (0, 2, 4, 6, 8, 10, 12, 24, 32, 48, 72 and 96 h) and urine samples (0, 1, 4, 8, 24, 32, 48, 72 and 96 h) were collected for analysis using liquid chromatography mass spectrometry. RESULTS: Maximum plasma concentrations (tmax ) were reached by the earliest detection point (2 h) after nebulisation (0.6-1.8 ng/mL), but was no longer detectable at 48 h. However, in one horse 0.1 ng/mL was found at 96 h after three consecutive readings of 0 ng/mL. The tmax in urine was reached by the earliest collection point (1 h) after nebulisation (3.2-23.8 ng/mL), but was no longer present in urine at 72 h in five horses, while detectable levels (0.1 ng/mL) were still present at 96 h in one horse. CONCLUSIONS: A single dose of 0.04 mg/kg of DSP administered as an aerosol through a FlexinebE2® mask was no longer detectable in blood at 48 h in six horses tested, but one horse returned a reading of 0.1 ng/mL at 96 h after having no detectable levels. Dexamethasone was not detectable in urine at 72 h in five horses but was detectable at a low concentration (0.1 ng/mL) at 96 h in one horse.

Removal of Dental Biofilms with an Ultrasonically Activated Water Stream.

Acidogenic bacteria within dental plaque biofilms are the causative agents of caries. Consequently, maintenance of a healthy oral environment with efficient biofilm removal strategies is important to limit caries, as well as halt progression to gingivitis and periodontitis. Recently, a novel cleaning device has been described using an ultrasonically activated stream (UAS) to generate a cavitation cloud of bubbles in a freely flowing water stream that has demonstrated the capacity to be effective at biofilm removal. In this study, UAS was evaluated for its ability to remove biofilms of the cariogenic pathogen Streptococcus mutans UA159, as well as Actinomyces naeslundii ATCC 12104 and Streptococcus oralis ATCC 9811, grown on machine-etched glass slides to generate a reproducible complex surface and artificial teeth from a typodont training model. Biofilm removal was assessed both visually and microscopically using high-speed videography, confocal scanning laser microscopy (CSLM), and scanning electron microscopy (SEM). Analysis by CSLM demonstrated a statistically significant 99.9% removal of S. mutans biofilms exposed to the UAS for 10 s, relative to both untreated control biofilms and biofilms exposed to the water stream alone without ultrasonic activation (P < 0.05). The water stream alone showed no statistically significant difference in removal compared with the untreated control (P = 0.24). High-speed videography demonstrated a rapid rate (151 mm(2) in 1 s) of biofilm removal. The UAS was also highly effective at S. mutans, A. naeslundii, and S. oralis biofilm removal from machine-etched glass and S. mutans from typodont surfaces with complex topography. Consequently, UAS technology represents a potentially effective method for biofilm removal and improved oral hygiene.

Purification of overexpressed gam gene protein from bacteriophage Mu by denaturation-renaturation techniques and a study of its DNA-binding properties.

Recombinant Mu gam gene protein (Mu GAM) synthesized in Escherichia coli accumulates in the form of insoluble inclusion bodies which, after cell lysis and low-speed centrifugation, can be recovered in the pellet fraction. This property was utilized in a purification procedure for Mu GAM based on guanidine hydrochloride denaturation-renaturation followed by a single DEAE-cellulose chromatographic step. The purified Mu GAM was shown by nitrocellulose-filter-binding experiments to bind with high affinity to linear double-stranded DNA and more weakly to supercoiled and single-stranded forms. Mu GAM protects linear DNA from degradation by a variety of exonucleases, but only weakly inhibits endonuclease activity. These results are in accord with a model of Mu GAM conferring protection from exonuclease activity by binding to the ends of DNA.

Identification of the bacteriophage Mu kil gene.

The kil gene encoded in bacteriophage Mu DNA was previously shown to reside between the end of the B gene at 4.3 kb and the EcoRI site at 5.1 kb from the left end. To precisely map the kil gene within this region, two series of BAL-31 deletion derivatives were created: one removed Mu DNA rightward from the Hpal site (4.2 kb) and the other removed Mu DNA leftward from the EcoRI site. The deleted Mu DNA was subcloned into the expression vector pUC19 under lac promoter control and tested for the expression of the killing function following IPTG induction. Using DNA sequencing analysis, the Mu DNA in Kil+ and Kil- clones was precisely determined, and the kil gene was mapped to the first open reading frame beyond the B gene. The expression of the kil gene was sufficient to induce dramatic morphological changes: cells became enlarged and predominantly spherical, reminiscent of the phenotype of certain cell mutants.

Localization of the gam gene of bacteriophage mu and characterisation of the gene product.

Using cloning techniques in conjunction with an in vitro assay for activity of the gam-coded protein (pgam), the gam gene has been located on a 930-bp fragment immediately to the right of an AccI site situated 5.75 kb from the left-hand end of the phage Mu genome. An analysis of the properties of pgam obtained from an overproducing clone indicates that it is a non-specific DNA-binding protein which interacts with linear duplex plasmid DNA having a variety of different termini and confers protection against exonuclease action (Gam function). It also stimulates the frequency with which linear plasmid DNA transforms Escherichia coli to antibiotic resistance (Sot function). The preliminary results reported here suggest that pgam is potentially a useful 'tool' in molecular biology, although the molecular details of pgam activity require further clarification.

Evidence for a conservative pathway of transposition of bacteriophage Mu.

During its lytic cycle bacteriophage Mu uses repeated transposition as a mode of DNA synthesis. These transpositional events are undoubtedly replicative, and presumably semi-conservative. In a Mu lysogen this type of transposition can start immediately after prophage induction. However, in an infective cycle the Mu genome (which is injected into the host cell as a linear molecule flanked by short random sequences of bacterial DNA) must first become integrated into the host chromosome. Little is known about how this occurs apart from the fact that the bacterial sequences at either end of the Mu genome are lost in the process. The integration is thus similar to a transposition event. In an attempt to determine whether this type of Mu transposition (between a linear donor molecule and a circular recipient) is also semi-conservative we have analysed the progeny phage arising from an infective cycle in which the parental DNA was heterozygous for a known genetic marker. The expectation is that if integration of the infecting Mu genome occurs by a single semi-conservative transpositional event then pure phage bursts should be produced as the genetic information on only one strand would be preserved throughout the lytic cycle. The experiments reported here do not support this expectation in that the infected cells yield mixed bursts, suggesting that Mu integration is a conservative, rather than a semi-conservative event.

Abnormal cointegrate structures mediated by gene B mutants of phage Mu: their implications with regard to gene function.

A system where the transposition of MupApl (a derivative of phage Mu carrying a determinant coding for ampicillin resistance) is followed from the small plasmid pML2 into the conjugative plasmid R388 has been used to investigate the influence on Mu transposition of B, an early Mu gene which is involved in normal phage DNA synthesis. In the absence of active B protein a low level (about 1% of normal) of transposition was detected. Roughly a third of these transpositional events was found to lead to the formation of cointegrate DNA structures which were shown to consist of R388, two complete copies of Mu and part only of pML2. The pML2 deletions vary in size but all those investigated appear to originate at an end of Mu. An explanation of these observations is proposed which envisages the B protein as part of the normal transposition complex.