Characterization of full-length recombinant human Proteoglycan 4 as an ocular surface boundary lubricant.

Proteoglycan 4 (PRG4, or lubricin) is a lubricating mucin-like glycoprotein recently discovered at the ocular surface, where it functions as a boundary lubricant and appears to play a protective role. Recent technological advances have enabled abundant expression of full-length recombinant human PRG4 (rhPRG4). The objectives of this study were to 1) biochemically characterize the gross structure and glycosylations of full-length rhPRG4, and 2) assess the ocular surface boundary lubricating ability of rhPRG4 at both human cornea-eyelid and human cornea-polydimethylsiloxane (PDMS) biointerfaces. rhPRG4 expressed by a Chinese hamster ovary cell line was characterized and compared to native bovine PRG4 by SDS-PAGE western blotting, and protein identity was assessed by tandem mass spectrometry (MS/MS). Human corneas were articulated against PDMS or human eyelids, at effective sliding velocities of 0.3-30 mm/s under physiological loads of ∼15 kPa, to assess and compare the ocular lubricating ability of rhPRG4 to PRG4. Samples were tested serially in PRG4, rhPRG4 (both 300 µg/ml), then saline. Western blotting indicated that rhPRG4 had immunoreactivity at the appropriate apparent molecular weight, and possessed O-linked glycosylation consistent with that of PRG4. rhPRG4 protein identity was confirmed by MS/MS. Both PRG4 and rhPRG4 significantly, and similarly, reduced friction compared to saline at both human cornea - PDMS and human cornea-eyelid biointerfaces. In conclusion, the rhPRG4 studied here demonstrated appropriate higher order structure, O-linked glycosylations, and ocular surface boundary lubricating. Purified rhPRG4 may have clinical utility as a topical treatment of dry eye disease or contact lens biomaterial coating to promote more comfortable wear.

Expert surgeon's quiet eye and slowing down: expertise differences in performance and quiet eye duration during identification and dissection of the recurrent laryngeal nerve.

BACKGROUND: Long quiet eye (QE) duration is central to expertise in sports, while cognitive "slowing down" has been identified as a perceptual skill possessed by skilled surgeons. Eye-tracking evidence is lacking about the relationship of QE duration to slowing down in surgeons. The aim of this study was to examine QE duration, hand movement time (MT), fixation location, and fixation duration in highly experienced (HE) and less experienced (LE) surgeons. METHODS: A mobile eye tracker and camera recorded coupled gaze and hand movements. Performance was quantified by blinded review. RESULTS: HE surgeons were rated higher than LE surgeons but did not differ in operating time or MT. HE and LE surgeons differed in fixation duration on the ligament of Berry during phases 1 and 2 and QE duration on the recurrent laryngeal nerve in phase 2. CONCLUSIONS: Long-duration fixation on the ligament of Berry and long-duration QE on the recurrent laryngeal nerve combined with no significant differences in MT provide empirical evidence that HE surgeons cognitively slow down more than LE surgeons during critical phases of the operation.

Transcription, translation, and function of lubricin, a boundary lubricant, at the ocular surface.

IMPORTANCE: Lubricin may be an important barrier to the development of corneal and conjunctival epitheliopathies that may occur in dry eye disease and contact lens wear. OBJECTIVE: To test the hypotheses that lubricin (ie, proteoglycan 4 [PRG4 ]), a boundary lubricant, is produced by ocular surface epithelia and acts to protect the cornea and conjunctiva against significant shear forces generated during an eyelid blink and that lubricin deficiency increases shear stress on the ocular surface and promotes corneal damage. DESIGN, SETTING, AND PARTICIPANTS: Human, porcine, and mouse tissues and cells were processed for molecular biological, immunohistochemical, and tribological studies, and wild-type and PRG4 knockout mice were evaluated for corneal damage. RESULTS: Our findings demonstrate that lubricin is transcribed and translated by corneal and conjunctival epithelial cells. Lubricin messenger RNA is also present in lacrimal and meibomian glands, as well as in a number of other tissues. Absence of lubricin in PRG4 knockout mice is associated with a significant increase in corneal fluorescein staining. Our studies also show that lubricin functions as an effective friction-lowering boundary lubricant at the human cornea-eyelid interface. This effect is specific and cannot be duplicated by the use of hyaluronate or bovine serum albumin solutions. CONCLUSIONS AND RELEVANCE: Our results show that lubricin is transcribed, translated, and expressed by ocular surface epithelia. Moreover, our findings demonstrate that lubricin presence significantly reduces friction between the cornea and conjunctiva and that lubricin deficiency may play a role in promoting corneal damage.

Dose-dependent and synergistic effects of proteoglycan 4 on boundary lubrication at a human cornea-polydimethylsiloxane biointerface.

OBJECTIVES: Proteoglycan 4 (PRG4), also known as lubricin, is a boundary lubricating mucin-like glycoprotein present on several tissue surfaces in the body. The objectives of this study were to (1) implement and characterize an in vitro boundary lubrication test at a human cornea-polydimethylsiloxane (PDMS) biointerface and (2) determine the dose-dependent and synergistic effects of PRG4, with hyaluronan (HA), on ocular surface boundary lubrication using this test. METHODS: Human corneas and model PDMS material were articulated against each other, at effective sliding velocities v(eff) between 0.3 and 30 mm/sec under physiologic loads of approximately 8 to 25 kPa. Samples were tested serially in (1) saline, PRG4 at 30, 100, 300 µg/mL resuspended in saline, then saline again or (2) saline, AQuify Comfort Eye Drops (containing 0.1% HA), 300 µg/mL PRG4 in saline, 300 µg/mL PRG4 in AQuify, then saline again. Both static and kinetic friction coefficients were calculated. RESULTS: PRG4 effectively lowered friction at the cornea-PDMS biointerface, both alone in a dose-dependent manner and in combination with HA. PRG4 reduced kinetic friction coefficients, <µ(kinetic, Neq)>, from approximately 0.30 in saline, to approximately 0.30, 0.24, and 0.17 in 30, 100, and 300 µg/mL PRG4, respectively. Values of <µ(kinetic, Neq)> in AQuify, approximately 0.32, were similar to those in saline; however, when combined with 300 µg/mL PRG4, values of <µ(kinetic, Neq)> were reduced to approximately 0.15. CONCLUSIONS: PRG4 functions as an effective ocular surface boundary lubricant, both alone in a dose-dependent manner and in combination with HA.

Influence of virtual reality training on the roadside crossing judgments of child pedestrians.

The roadside crossing judgments of children aged 7, 9, and 11 years were assessed relative to controls before and after training with a computer-simulated traffic environment. Trained children crossed more quickly, and their estimated crossing times became better aligned with actual crossing times. They crossed more promptly, missed fewer safe opportunities to cross, accepted smaller traffic gaps without increasing the number of risky crossings, and showed better conceptual understanding of the factors to be considered when making crossing judgments. All age groups improved to the same extent, and there was no deterioration when children were retested 8 months later. The results are discussed in relation to theoretical arguments concerning the extent to which children's pedestrian judgments are amenable to training.

The effects of adult guidance and peer discussion on the development of children's representations: evidence from the training of pedestrian skills.

It was hypothesized that practical training is effective in improving children's pedestrian skills because adult scaffolding and peer discussion during training specifically promote E3 level representation (linguistically-encoded, experientially-grounded, generalizable knowledge), as defined by Karmiloff-Smith's (1992) representational redescription (RR) model. Two studies were conducted to examine in detail the impact of this social input in the context of simulation-based training in roadside search skills. A group of 5-8-year-olds were pre-tested on ability to detect relevant road-crossing features. They then participated in four training sessions designed to promote attunement to these, under peer discussion versus adult guidance conditions (Study 1), and adult-child versus adult-group conditions (Study 2). Performance at post-test was compared with that of controls who underwent no training. Study 1 found that children in the adult guidance condition improved significantly more than those in the peer discussion or control conditions, and this improvement was directly attributable to appropriation of E3 level representations from adult dialogue. Study 2 found that progress was greater still when adult scaffolding was supplemented by peer discussion, with E3 level representation attributable to the children's exploration of conflicting ideas. The implications of these findings for the RR model and for practical road safety education are discussed.