In silico analysis enabling informed design for genome editing in medicinal cannabis; gene families and variant characterisation.

BACKGROUND: Cannabis has been used worldwide for centuries for industrial, recreational and medicinal use, however, to date no successful attempts at editing genes involved in cannabinoid biosynthesis have been reported. This study proposes and develops an in silico best practices approach for the design and implementation of genome editing technologies in cannabis to target all genes involved in cannabinoid biosynthesis. RESULTS: A large dataset of reference genomes was accessed and mined to determine copy number variation and associated SNP variants for optimum target edit sites for genotype independent editing. Copy number variance and highly polymorphic gene sequences exist in the genome making genome editing using CRISPR, Zinc Fingers and TALENs technically difficult. Evaluation of allele or additional gene copies was determined through nucleotide and amino acid alignments with comparative sequence analysis performed. From determined gene copy number and presence of SNPs, multiple online CRISPR design tools were used to design sgRNA targeting every gene, accompanying allele and homologs throughout all involved pathways to create knockouts for further investigation. Universal sgRNA were designed for highly homologous sequences using MultiTargeter and visualised using Sequencher, creating unique sgRNA avoiding SNP and shared nucleotide locations targeting optimal edit sites. CONCLUSIONS: Using this framework, the approach has wider applications to all plant species regardless of ploidy number or highly homologous gene sequences. SIGNIFICANCE STATEMENT: Using this framework, a best-practice approach to genome editing is possible in all plant species, including cannabis, delivering a comprehensive in silico evaluation of the cannabinoid pathway diversity from a large set of whole genome sequences. Identification of SNP variants across all genes could improve genome editing potentially leading to novel applications across multiple disciplines, including agriculture and medicine.

Frontal ostium neo-osteogenesis and restenosis after modified endoscopic Lothrop procedure in an animal model.

All patients who undergo a modified endoscopic Lothrop procedure have postoperative narrowing of the enlarged frontal ostium. The aim of this study is to evaluate neo-osteogenesis and restenosis of the frontal ostium and its effect on mucociliary clearance. Fourteen sheep underwent an endoscopic modified Lothrop procedure. Pre- and postoperative nuclear medicine gamma scintigraphy of the frontal sinuses was performed. The sizes of the frontal ostia were measured and biopsies taken from the bone of the frontal ostium. Histological evidence of new bone formation was found in 56% of biopsies. The average preoperative mucociliary clearance half times (T1/2) at 15 and 30 min were 70 and 74 min, respectively, and postoperatively were 50 and 67 min. There was a non-significant trend towards poorer clearance in sinuses with neo-osteogenesis. The average size of the frontal ostium decreased by 28%. There was no relationship between the size of the ostium and neo-osteogenesis. Neo-osteogenesis was seen in 56% of biopsies with a 28% reduction in size of the frontal ostium after 224 days. Mucociliary clearance did not alter significantly.

The effect of the modified endoscopic Lothrop procedure on the mucociliary clearance of the frontal sinus in an animal model.

BACKGROUND: The surgical management of recalcitrant frontal sinus disease has been a dilemma for otolaryngologists for many years. Although the osteoplastic flap with obliteration has been the gold standard of treatment for years, the modified endoscopic Lothrop (MEL) procedure recently has been advocated as an alternative. However, little is known about the effect of this procedure on the mucociliary drainage of the frontal sinuses postoperatively and this animal study addresses this issue. METHODS: Fourteen sheep underwent the MEL procedure. The sheep were randomized regarding the use of postoperative irrigation via minitrephines. Each sheep had a nuclear medicine gamma-scintigraphy frontal sinus clearance study via minitrephines performed on each frontal sinus preoperatively and then 3 months postoperatively. Then, the results of these studies were compared. RESULTS: The scans revealed a trend toward faster clearance times postoperatively. However, this decrease was not statistically significant. Importantly, there was no trend or significant increase in clearance times postoperatively. Also, the use of postoperative irrigation was associated with a nonsignificant trend toward faster clearance times postoperatively. CONCLUSION: The MEL procedure has no adverse effects on the mucociliary clearance of the frontal sinus at 3 months postoperatively. Irrigation of the frontal sinus in the immediate postoperative period showed a trend toward improved postoperative mucociliary function at 3 months.

Polypharmacy in a headache centre population.

In this paper we report the prevalence of self-medication and polypharmacy in our patient population at the John R. Graham Headache Centre at the Faulkner Hospital, Boston. One hundred fifty patients were interviewed when they called the Headache Centre and spoke to a triage nurse. A significantly higher number of women than men called in for triage counselling. Sixty-nine percent of the patients reported taking non-Headache Centre medications, either prescribed or over the counter (OTC); 39% of the patients took pain medications. Thirty-one different medications were named by patients for a mix of diagnoses.