Hydrogen Peroxide-Based Fluorometric Assay for Real-Time Monitoring of SAM-Dependent Methyltransferases.

Methylated chemicals are widely used as key intermediates for the syntheses of pharmaceuticals, fragrances, flavors, biofuels and plastics. In nature, the process of methylation is commonly undertaken by a super-family of S-adenosyl methionine-dependent enzymes known as methyltransferases. Herein, we describe a novel high throughput enzyme-coupled assay for determining methyltransferase activites. Adenosylhomocysteine nucleosidase, xanthine oxidase, and horseradish peroxidase enzymes were shown to function in tandem to generate a fluorescence signal in the presence of S-adenosyl-L-homocysteine and Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine). Since S-adenosyl-L-homocysteine is a key by-product of reactions catalyzed by S-adenosyl methionine-dependent methyltransferases, the coupling enzymes were used to assess the activities of EcoRI methyltransferase and a salicylic acid methyltransferase from Clarkia breweri in the presence of S-adenosyl methionine. For the EcoRI methyltransferase, the assay was sensitive enough to allow the monitoring of DNA methylation in the nanomolar range. In the case of the salicylic acid methyltransferase, detectable activity was observed for several substrates including salicylic acid, benzoic acid, 3-hydroxybenzoic acid, and vanillic acid. Additionally, the de novo synthesis of the relatively expensive and unstable cosubstrate, S-adenosyl methionine, catalyzed by methionine adenosyltransferase could be incorporated within the assay. Overall, the assay offers an excellent level of sensitivity that permits continuous and reliable monitoring of methyltransferase activities. We anticipate this assay will serve as a useful bioanalytical tool for the rapid screening of S-adenosyl methionine-dependent methyltransferase activities.

Merkel cell carcinoma of the eyelid: management and prognosis.

IMPORTANCE: The literature on Merkel cell carcinoma (MCC) of the eyelid remains scarce, and there has yet to be a study using the most up-to-date TNM staging system for this rare but aggressive tumor. OBJECTIVE: To analyze the TNM stage, management, and outcomes of patients with MCC of the eyelid. DESIGN, SETTING, AND PARTICIPANTS: Retrospective case series of 21 patients from 5 tertiary referral centers in the United Kingdom and Australia with primary MCC of the eyelid presenting at a median age of 77 years, with median follow-up of 54 months. Tumors were staged according to the American Joint Committee on Cancer, 7th edition, TNM criteria for eyelid carcinoma and MCC. MAIN OUTCOMES AND MEASURES: TNM stage, treatment modalities, and clinical outcome. RESULTS: The eyelid carcinoma TNM stages were T2aN0M0 for 5 patients, T2bN0M0 for 7 patients, T3aN0M0 for 4 patients, T3bN0M0 for 3 patients, T2bN1M0 for 1 patient, and T3aN1M0 for 1 patient. The MCC TNM stages were T1N0M0 for 12 patients, T2N0M0 for 7 patients, T1N1M0 for 1 patient, and T2N1M0 for 1 patient. One patient had a sentinel lymph node biopsy, and 8 patients underwent head/neck imaging. Eighteen patients underwent a wide local excision, 12 with a paraffin section and 6 with a frozen section. Two patients underwent Mohs surgery, 1 of whom required an orbital exenteration. Twelve patients (57%) received adjuvant radiotherapy, and 2 patients received chemotherapy. The local recurrence rate was 10%, the regional nodal recurrence rate was 10%, and the distant metastatic recurrence rate was 19%. The lowest T category tumor metastasizing to both regional nodes and distant locations was a T2a (eyelid TNM)/T1 (Merkel TNM) tumor measuring 8 mm. Two patients with T3a (eyelid TNM)/T2 (Merkel TNM) tumors died of metastatic MCC. CONCLUSIONS AND RELEVANCE: The majority of patients with MCC of the eyelid present with localized eyelid disease of T category T2 (eyelid TNM)/T1 (Merkel TNM). A wide local excision with margin control remains the mainstay of treatment, whereas the use of radiotherapy is institution specific. Tumors with a low T category are associated with regional nodal and distant metastatic disease. It may therefore be reasonable to consider a sentinel lymph node biopsy or strict regional lymph node surveillance for all MCCs of the eyelid, regardless of T category or size.

Monocanalicular versus bicanalicular intubation in external dacryocystorhinostomy for primary acquired nasolacrimal duct obstruction.

PURPOSE: Monocanalicular intubation offers potential advantages over bicanalicular intubation including ease of removal and use of only one canaliculus. Recent reports suggest equal outcomes with both types of tubes for nasolacrimal intubation. This study aimed to evaluate the outcome of monocanalicular intubation during external dacryocystorhinostomy. METHODS: Fifty-seven lacrimal systems of 54 adults with primary acquired nasolacrimal duct obstruction were prospectively randomized into two cohorts for either bicanalicular intubation (n = 29) or monocanalicular intubation (n = 28) during external dacryocystorhinostomy. All subjects had their symptoms evaluated subjectively according to a modified Munk scale and objectively by the dye disappearance test along with probing and irrigation, pre- and post-operatively. Complete success was defined as a total disappearance of symptoms, partial success as an improvement with some residual symptoms, and failure as an absence of improvement or worsening of symptoms at the last follow-up. Patients with concurrent lid or ocular pathology were excluded. RESULTS: Bicanalicular subjects had a significantly higher complete success rate (21/29, 72.4%) compared to the monocanalicular group (12/28, 42.9%) (p = 0.03). Complications included 3 slit punctuae and 2 early tube removals with bicanalicular intubation; 6 temporary superficial punctate keratopathies, 1 punctal stenosis and 4 premature tube losses occurred with monocanalicular intubation. CONCLUSIONS: Patients with monocanalicular intubation during external dacryocystorhinostomy had a significantly lower success rate than patients with bicanalicular intubation in the treatment of nasolacrimal duct obstruction.

The long and winding road: contact lens-induced ptosis.

Intrapalpebral migration is a rare complication of hard contact lens use. We report a case of a 52-year-old man who presented with right upper eyelid ptosis and requested cosmetic correction. He had no ocular history apart from myopia, which was corrected with the use of hard contact lenses. Prior to the ptosis he recalled losing his right contact lens during a bout of sneezing. He replaced the missing lens and continued to wear contact lenses as usual. After careful clinical examination, an ipsilateral small, mobile eyelid mass was detected, and orbital MRI verified the suspected presence of a hard contact lens. Following surgical removal and ptosis correction, the patient made a full recovery. The phenomenon of hard contact lens migration in the periocular tissues has been reported in the literature before, but most patients experienced symptoms of variable severity. In this patient, however, there were no symptoms to indicate the presence of a contact lens. This case highlights the importance of including intrapalpebral contact lens migration in the differential diagnosis of an eyelid mass or ptosis in patients.

A novel approach to lacrimal sac dissection in dacryocystorhinostomy.

The use of membrane-peeling vitreoretinal forceps to facilitate lacrimal sac dissection in external dacryocystorhinostomy is demonstrated. The closed vitreoretinal forceps are advanced via the upper punctum and canaliculus, through to the lacrimal sac. They are then opened maximally providing two points of elevation. This maneuver allows for easier identification of the plane of dissection during lacrimal flap creation. It is especially useful with complex anatomy and for junior trainees in helping to orient the dissection. No tissue or instrument damage has been noted, and because many ophthalmic units have a vitreoretinal service, the forceps may be readily available at no extra cost.

Camera adapter for anterior segment slitlamp photography.

We describe an original, affordable and readily accessible slit lamp camera adapter. This is easily assembled from parts already available in most eye units, allowing ready access to anterior segment photography, using both digital and analog cameras.