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The link between form and function is key to understanding the evolution of unique and/or extreme morphologies. Amblypygids, or whip spiders, are arachnids that often have highly elongated spined pedipalps. These limbs are used to strike at, and secure, prey before processing by the chelicerae. Amblypygi pedipalps are multifunctional, however, being used in courtship and contest, and vary greatly in form between species. Increased pedipalp length may improve performance during prey capture, but length could also be influenced by factors including territorial contest and sexual selection. Here, for the first time, we used high-speed videography and manual tracking to investigate kinematic differences in prey capture between amblypygid species. Across six morphologically diverse species, spanning four genera and two families, we created a total dataset of 86 trials (9-20 per species). Prey capture kinematics varied considerably between species, with differences being expressed in pedipalp joint angle ranges. In particular, maximum reach ratio did not remain constant with total pedipalp length, as geometric scaling would predict, but decreased with longer pedipalps. This suggests that taxa with the most elongated pedipalps do not deploy their potential length advantage to proportionally increase reach. Therefore, a simple mechanical explanation of increased reach does not sufficiently explain pedipalp elongation. We propose other factors to help explain this phenomenon, such as social interactions or sexual selection, which would produce an evolutionary trade-off in pedipalp length between prey capture performance and other behavioural and/or anatomical pressures., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Sexual differences in size and shape are common across the animal kingdom. The study of sexual dimorphism (SD) can provide insight into the sexual- and natural-selection pressures experienced by males and females in different species. Arachnids are diverse, comprising over 100,000 species, and exhibit some of the more extreme forms of SD in the animal kingdom, with the males and females of some species differing dramatically in body shape and/or size. Despite this, research on arachnid SD has primarily focused on specific clades as opposed to observing traits across arachnid orders, the smallest of which have received comparatively little attention. This review provides an overview of the research to date on the trends and potential evolutionary drivers for SD and sexual size dimorphism (SSD) in individual arachnid orders, and across arachnids as a whole. The most common trends across Arachnida are female-biased SSD in total body size, male-biased SSD in relative leg length and SD in pedipalp length and shape. However, the evolution of sexually dimorphic traits within the group is difficult to elucidate due to uncertainty in arachnid phylogenetic relationships. Based on the dataset we have gathered here, we highlight gaps in our current understanding and suggest areas for future research., Competing Interests: The authors declare that they have no competing interests.

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 Eco RI methyltransferase and a salicylic acid methyltransferase from Clarkia breweri in the presence of S-adenosyl methionine. For the Eco RI 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.

Sphingolipids (SLs) are ubiquitous elements in eukaryotic membranes and are also found in some bacterial and viral species. As well as playing an integral structural role, SLs also act as potent signaling molecules involved in numerous cellular pathways and have been linked to many human diseases. A central SL signaling molecule is sphingosine-1-phosphate (S1P), whose breakdown is catalyzed by S1P lyase (S1PL), a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the cleavage of S1P to (2E)-hexadecenal (2E-HEX) and phosphoethanolamine. Here, we show that the pathogenic bacterium, Burkholderia pseudomallei K96243, encodes two homologous proteins (S1PL2021 and S1PL2025) that display moderate sequence identity to known eukaryotic and prokaryotic S1PLs. Using an established MS-based methodology, we show that recombinant S1PL2021 is catalytically active. We also used recombinant human fatty aldehyde dehydrogenase to develop a spectrophotometric enzyme-coupled assay to detect 2E-HEX formation and measure the kinetic constants of the two B. pseudomallei S1PL isoforms. Furthermore, we determined the X-ray crystal structure of the PLP-bound form of S1PL2021 at 2.1 Å resolution revealing that the enzyme displays a conserved structural fold and active site architecture comparable with known S1PLs. The combined data suggest that B. pseudomallei has the potential to degrade host SLs in a S1PL-dependent manner., (Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.)

Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, plays a critical role in the orchestration of immune responses. S1P levels within the mammalian host are tightly regulated, in part through the activity of S1P lyase (S1PL) which catalyses its irreversible degradation. Herein, we describe the identification and characterization of secreted S1PL orthologues encoded by the facultative intracellular bacteria Burkholderia pseudomallei and Burkholderia thailandensis. These bacterial orthologues exhibited S1PL enzymatic activity, functionally complemented an S1PL-deficient yeast strain and conferred resistance to the antimicrobial sphingolipid D-erythro-sphingosine. We report that secretion of these bacterial S1PLs is pH-dependent, and is observed during intracellular infection. S1PL-deficient mutants displayed impaired intracellular replication in murine macrophages (associated with an inability to evade the maturing phagosome) and were significantly attenuated in murine and larval infection models. Furthermore, treatment of Burkholderia-infected macrophages with either S1P or a selective agonist of S1P receptor 1 enhanced bacterial colocalisation with LAMP-1 and reduced their intracellular survival. In summary, our studies confirm bacterial-encoded S1PL as a critical virulence determinant of B. pseudomallei and B. thailandensis, further highlighting the pivotal role of S1P in host-pathogen interactions. In addition, our data suggest that S1P pathway modulators have potential for the treatment of intracellular infection., (© 2016 John Wiley & Sons Ltd.)

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.

Rationale: The preterm diaphragm is structurally and functionally immature, potentially contributing to an increased risk of respiratory distress and failure. We investigated developmental changes in contractile function and susceptibility to fatigue of the costal diaphragm in the fetal lamb to understand factors contributing to the risk of developing diaphragm dysfunction and respiratory disorders. We hypothesized that the functional capacity of the diaphragm will vary with maturational stage as will its susceptibility to fatigue., Methods: Lambs were studied at 75, 100, 125, 145, 154, 168, and 200 days postconceptional age (term = 147 days). Lambs were euthanized (sodium pentobarbitone, 100 mg/kg) either at delivery or immediately prior to post-mortem for postnatal lambs. Contractile function was assessed on longitudinal strips of intact muscle fibers and the remaining tissue frozen in liquid nitrogen for analysis of myosin heavy chain (MHC) mRNA expression and protein content., Results: Fetal development of diaphragm function was characterized by a significant increase in maximum specific force, increased susceptibility to fatigue, reduced twitch contraction times, and a progressive increase in MHCI and MHCII protein content. Postnatally, there was a progressive decrease in the susceptibility to fatigue that coincided with an increase in the MHC I:II protein ratio., Conclusion: These data indicate that the functional capacity of the diaphragm varies with maturational age and may be an important determinant of the susceptibility to preterm respiratory failure., (Copyright © 2013 Wiley Periodicals, Inc.)

Increasing positive end-expiratory pressure (PEEP) is advocated to recruit alveoli during high-frequency jet ventilation (HFJV), but its effect on cardiopulmonary physiology and lung injury is poorly documented. We hypothesized that high PEEP would recruit alveoli and reduce lung injury but compromise pulmonary blood flow (PBF). Preterm lambs of anesthetized ewes were instrumented, intubated, and delivered by cesarean section after instillation of surfactant. HFJV was commenced with a PEEP of 5 cm H2O. Lambs were allocated randomly at delivery to remain on constant PEEP (PEEPconst, n = 6) or to recruitment via stepwise adjustments in PEEP (PEEPadj, n = 6) to 12 cm H2O then back to 8 cm H2O over the initial 60 min. PBF was measured continuously while ventilatory parameters and arterial blood gases were measured at intervals. At postmortem, in situ pressure-volume deflation curves were recorded, and bronchoalveolar lavage fluid and lung tissue were obtained to assess inflammation. PEEPadj lambs had lower pressure amplitude, fractional inspired oxygen concentration, oxygenation index, and PBF and more compliant lungs. Inflammatory markers were lower in the PEEPadj group. Adjusted PEEP during HFJV improves oxygenation and lung compliance and reduces ventilator requirements despite reducing pulmonary perfusion.

Increases in positive end-expiratory pressure (PEEP) improve arterial oxygenation in preterm infants, but the effects on cardiopulmonary hemodynamics are understood poorly. We aimed to determine the effect of increased PEEP on cardiopulmonary hemodynamics and to compare measurements from indwelling flow probes with Doppler echocardiography. Preterm lambs (129 +/- 1 days) were ventilated initially with a tidal volume of 7 ml/kg and 4 cmH(2)O of PEEP. In ramp lambs (n = 7), PEEP was increased by 2-cmH(2)O increments to 10 cmH(2)O and then in decrements back to 4 cmH(2)O. PEEP was unchanged in controls (n = 6). Doppler echocardiographic flow measurements in the left pulmonary artery (LPA) and ductus arteriosus (DA) were correlated with flow probe measurements. Compared with controls, high PEEP reduced LPA flow from baseline (10-cmH(2)O PEEP: 43 +/- 8% vs. control: 83 +/- 21%; P = 0.029). High PEEP increased the proportion of right-to-left (R-L) shunting through the DA, with a trend to an increased oxygenation index compared with controls (oxygenation index: 44.5 +/- 13.5 at 10-cmH(2)O PEEP vs. 19.4 +/- 4.5 in controls; P = 0.07). Increasing PEEP decreased heart rate (17 beats/min; P = 0.03) and tended to lower systolic arterial pressure (5.0 mmHg; P = 0.052) compared with controls. Doppler echocardiography measurement of LPA flows correlated strongly with indwelling flow probe (r(2) = 0.73, P < 0.001), except during highly turbulent flows. Increases in PEEP have significant cardiopulmonary consequences in preterm lambs, including reduced LPA flow and increased R-L shunt through the DA. These changes are likely due to the concomitant increase in downstream pulmonary vascular resistance and increased cardiovascular constraint induced by PEEP.

The ST2 gene is a member of the interleukin-1 receptor family and is located on chromosome 2q12, an area of the genome that has been associated with asthma. The soluble product of the ST2 gene, serum ST2 (sST2), has previously been shown to be elevated in adult asthmatic patients. This study investigated the potential role of ST2 in children with acute asthma. Children aged 2-16 years (n = 186) were recruited on presentation with acute asthma in the emergency department. Blood was obtained on presentation and during convalescence. Variables assessed included sST2 levels, a comprehensive assembly of clinical parameters and two polymorphisms in the ST2 gene, -26999G/A, located in the distal promoter region, and ala78glu polymorphism, on exon 3. The A allele of the -26999G/A polymorphism occurred more frequently in asthmatics compared with an unselected control group (P = 0.031). Serum ST2 levels were substantially higher during acute asthma compared with levels after the attack: 0.29 ng/ml (95% confidence interval: 0.23-0.36) and 0.14 ng/ml (0.12-0.17), respectively (P = 0.001) and were inversely related to eosinophil counts during an acute asthma attack (P = 0.002). The -26999AA genotype, as well as the AC haplotype, was associated with asthma severity scores (P = 0.05 and 0.02) compared with the -26999GA and GG genotypes. Serum ST2 levels were not associated with any of the studied genotypes or haplotypes. The observed associations of ST2 genotypes and haplotypes with acute asthma and asthma severity scores as well as the phenotypic differences associated with ST2 polymorphisms suggest that ST2 may play a role in the pathophysiology of asthma.

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.

Objective: We have shown previously that cutting or loading articular cartilage resulted in a fibroblast growth factor-2 (FGF-2) dependent activation of the extracellularly regulated kinase (ERK), and induction of a number of chondrocyte regulatory proteins including tissue inhibitor of metalloproteinase-1 and matrix metalloproteinases 1 and 3. An extracellular matrix-bound pool of FGF-2 was apparent, which could be liberated from the tissue by heparitinase (Vincent et al., Proc Natl Acad Sci U S A 2002;99(12):8259-64, Vincent et al., Arthritis Rheum 2004 Feb;50(2):526-33). Our objectives were to determine where FGF-2 was stored in articular cartilage, to which proteoglycan it was bound, and to elucidate its role in chondrocyte mechanotransduction., Methods: Immunohistochemistry and confocal microscopy were used to localise FGF-2 in the tissue. In vitro binding studies were performed using IASYS surface plasmon resonance. To study the role of pericellular FGF-2 in mechanotransduction cartilage explants or articular chondrocytes encapsulated in alginate were loaded using an in house loading rig. The loading response was assessed by the activation of ERK, in the presence or absence of a specific FGFR inhibitor., Results: Here we have identified perlecan as the heparan sulphate proteoglycan that sequesters FGF-2 in articular cartilage. Perlecan and FGF-2 co-localised within the type VI collagen-rich pericellular matrix of porcine and human articular cartilage. Chondrocytes encapsulated in alginate were able to accumulate pericellular perlecan and FGF-2 in culture, and deliver an FGF-dependent activation of ERK when loaded., Conclusion: Loading-induced ERK activation was dependent upon the presence and concentration of pericellular FGF-2, suggesting a functional role for this matrix-bound growth factor in chondrocyte mechanotransduction.

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.

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.

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.

Aims: To study the management of the orbital cysts present in a group of patients with anophthalmos and microphthalmos., Methods: A retrospective study of 34 patients (40 orbits) treated for orbital cyst associated with microphthalmos and anophthalmos., Results: The two largest treatment groups comprised 17 orbits (42.5%) where the cyst was removed surgically and 17 orbits (42.5%) where the cyst was retained and conformers were used. The remaining cases comprised two orbits (5%) where the cyst was aspirated initially; two orbits (5%) with large cysts which will need to be excised after further orbital growth; one orbit (2.5%) in which a silicone expander was used initially, and one orbit (2.5%) in which a mildly microphthalmic eye had some vision and was monitored but required no surgery., Conclusion: In this study 33 out of 34 patients had a good cosmetic result which illustrates that the orbital cyst in microphthalmos or anophthalmos performs a useful role in socket expansion and that the majority of patients with this condition can expect a good cosmetic outcome.

Objective: The purpose of the study was to review the surgical management of postherpetic lacrimal obstruction., Design: Retrospective noncomparative case series., Participants: One hundred sixty patients (111 female, 49 male) with a mean age at presentation of 31 years. All had a history typical of primary herpes simplex blepharoconjunctivitis., Intervention: Open lacrimal surgery was performed on 158 patients (171 eyes), of whom 99 patients (108 eyes) had undergone no surgery before being seen at Moorfields. The most common primary procedure was dacryocystorhinostomy (DCR) with anterograde or retrograde intubation (94 eyes, 54%), and primary placement of a Lester Jones tube (conjunctivo-DCR) was performed in 56 eyes (32%). A secondary procedure was required in 43 eyes (26%), the most common being closed placement of a Lester Jones tube (40 eyes)., Main Outcome Measures: Persistent symptoms of impaired lacrimal drainage and need for additional surgery., Results: Reduction of epiphora was good or complete in 171/173 eyes (98%)., Conclusions: This study shows that there is a justification for procedures that use any remaining unaffected portion of canaliculi, such as DCR, with anterograde or retrograde intubation, as well as the more commonly used Lester Jones tube.

Aims: To analyse bone fragments from rhinostomies of patients undergoing revisional dacryocystorhinostomy, looking for evidence of new bone formation., Methods: 14 consecutive patients undergoing secondary lacrimal surgery were included in this study. In each case the existing rhinostomy was enlarged with bone punches, care being taken to use the punches with the jaws cutting perpendicularly to the edge of the rhinostomy, to allow accurate orientation of the specimens. The fragments were examined histologically for evidence of new bone formation., Results: Histological sections showed fragments of bone with variable fibrosis at the edge of the rhinostomy. There was evidence of only very little new bone formation., Conclusion: This study has clearly shown that, at the edge of a rhinostomy, healing is predominantly by fibrosis and there is only very limited new bone formation.

Objective: To determine the incidence of ocular injuries associated with superglue., Methods: A retrospective review of patients attending an accident and emergency department over a 12 month period with ocular superglue injuries., Results: 14 patients who suffered ocular injuries due to superglue were identified. No patients had long term complications from their injury. Management is discussed., Conclusions: Superglue eye injuries do not appear to cause long term morbidity.

A tonometer prism holder is presented herein that can be quickly constructed from a readily available eyedrops bottle. The advantage of the stand is that only the tip of the tonometer prism is immersed in disinfectant, preventing damage to the proximal end of the prism. Consistent sterilization is ensured by renewing the disinfectant around the tonometer tip from an adjoining reservoir after each use. The stand is also leakproof, and if it is accidentally knocked over, disinfectant will not spill out.

Saliva was collected from six healthy young men at hourly intervals at sea level and after 1-2, 8-9 and 15-16 days at 4450 m on Mount Kenya for measurement of aldosterone (SA) and glucocorticoid (SGC, cortisol + cortisone) concentrations. Blood samples were collected simultaneously with some of the saliva samples and analysis of these showed that plasma and saliva concentrations of aldosterone and glucocorticoids were highly correlated (r = 0.91 and 0.75 respectively; p less than 0.01 for both hormones). Mean SA for the group was reduced to approximately 50% of the sea-level value (p less than 0.05) by the time the first saliva samples were collected at altitude, and remained at this depressed level throughout the 2-week period on Mount Kenya, although there was considerable inter-subject variation. SGC concentration also tended to be lower on Mount Kenya than at sea level. Though SA was lower throughout the day at altitude compared to sea level, the principal difference in the temporal pattern of SA was the reduction or complete absence of the marked rise in SA that normally occurs in the first few hours after rising. SA and SGC responses to exercise, which consisted of stepping on and off and 0.4-m high stool 60 times/min for 25 min, were assessed at sea level and after various periods at 4450 m.(ABSTRACT TRUNCATED AT 250 WORDS)

The Patterns of Care Study, begun in 1973, carried out its second outcome survey from December 1978 through April 1979. The review of patients with epidermoid carcinomas of the anterior two-thirds of tongue and floor of mouth included patients treated between January 1973 and June 1975 so that a minimum follow-up period of 2 years was evaluable. Records of 434 patients from 96 facilities were available for analysis, although not every patient could be evaluated for every variable. Almost three-fourths (320) were treated with radiation alone. In these data, four patient and disease characteristics were strongly related to recurrence rates including stage at presentation, deep ulceration, deep infiltration, and age. Treatment variables significantly affecting recurrence included use of interstitial therapy in Stages I and II, use of surgery in Stages III and IV, and a facility's best equipment.