Path Sampling Methods for Enzymatic Quantum Particle Transfer Reactions.

The mechanisms of enzymatic reactions are studied via a host of computational techniques. While previous methods have been used successfully, many fail to incorporate the full dynamical properties of enzymatic systems. This can lead to misleading results in cases where enzyme motion plays a significant role in the reaction coordinate, which is especially relevant in particle transfer reactions where nuclear tunneling may occur. In this chapter, we outline previous methods, as well as discuss newly developed dynamical methods to interrogate mechanisms of enzymatic particle transfer reactions. These new methods allow for the calculation of free energy barriers and kinetic isotope effects (KIEs) with the incorporation of quantum effects through centroid molecular dynamics (CMD) and the full complement of enzyme dynamics through transition path sampling (TPS). Recent work, summarized in this chapter, applied the method for calculation of free energy barriers to reaction in lactate dehydrogenase (LDH) and yeast alcohol dehydrogenase (YADH). We found that tunneling plays an insignificant role in YADH but plays a more significant role in LDH, though not dominant over classical transfer. Additionally, we summarize the application of a TPS algorithm for the calculation of reaction rates in tandem with CMD to calculate the primary H/D KIE of YADH from first principles. We found that the computationally obtained KIE is within the margin of error of experimentally determined KIEs and corresponds to the KIE of particle transfer in the enzyme. These methods provide new ways to investigate enzyme mechanism with the inclusion of protein and quantum dynamics.

Evaluation of the motion of surgical instruments during intraocular surgery.

PURPOSE: Robot assistance in ocular microsurgery could improve precision, dexterity, save time or prevent complications by task automation, and provide access to ocular surgery in undeserved countries by teleoperation. However, to design robotic devices, the range of motion of surgical instruments needs to be precisely quantified. METHODS: An electromagnetic tracking system was developed for intraocular surgery in order to quantify the movements of ophthalmic surgeons. Kinematics of surgical steps during phacoemulsification and pars plana vitrectomy procedures were determined by measuring the maximum translation and angular range of motion of intraocular surgical tools in the three planes. CONCLUSION: Important variations in amplitudes of rotation and translation were measured between both hands and between surgical tasks. These parameters may be used to develop a robotic intraocular surgical system or to improve training.

'The Microhand': a new concept of micro-forceps for ocular robotic surgery.

PURPOSE: To test the feasibility of retinal manipulations using a new micromanipulator (Microhand) for ocular robotic microsurgery. METHODS: Pneumatically actuated four-finger microhands were developed at UCLA with micro electromechanical systems (MEMS) technology to mimic a human hand for small object manipulation. Microhands with four 4 mm finger lengths were used for this study to lift caliper weights and fresh retinal tissue of porcine cadaver eyes to find the maximum force at a given pressure and feasibility of the microhands for retinal manipulation in real surgery. RESULTS: A full closure of the microhand used for caliper weight lifting was achieved under 65 psi (448 kPa) of air pressure. The four-fingered microhand was able to develop about 20 mN of total lifting force and 5 mN per finger at 80 psi (551 kPa), and was strong enough to displace and lift the retina of pig eyes. CONCLUSIONS: The microhand is able to apply calibrated forces to ocular tissues and is suitable for ocular microsurgical procedures. This new tool would be useful in the development of robotic microsurgery.

Robotic microsurgery: corneal transplantation.

BACKGROUND: Robotic ocular microsurgery including corneal suturing has been proven to be feasible in porcine eyes. AIM: To determine whether or not bimanual teleoperated robotic penetrating keratoplasty (PK) can be performed in porcine and human eyes. METHODS: Three arms of the da Vinci surgical robot were loaded with a dual-channel video and two, 360 degrees -rotating, 8 mm, wrested-end effector instruments and placed over porcine eyes or over a human cadaver head. The surgeon remotely performed mechanical trephination, cardinal sutures, continuous 10.0 nylon sutures and suture adjustments on both eyes. The procedures were documented with still and video photography. RESULTS: Using the da Vinci robot, penetrating keratoplasty procedures were successfully performed on both porcine eyes and human eyes in natural anatomical conditions. The precise placement of continuous sutures was facilitated by the wrested-end forceps. Orbital rims and nose did not limit surgical motions. CONCLUSION: Teleoperated robotic penetrating keratoplasty is technically feasible in humans. Further studies are pending to implement the procedure with femtosecond laser and other automated steps.

25-Gauge vitrectomy for paediatric vitreoretinal conditions.

UNLABELLED: This is a retrospective study of 56 eyes of 49 children undergoing vitrectomy with 25-gauge instrumentation. There were no cases of endophthalmitis, wound leaks or hypotony requiring treatment. A modified approach in which the conjunctiva and sclera is sutured was used for young babies requiring a pars plicata approach. OBJECTIVE: To evaluate the feasibility and safety of 25-gauge vitrectomy for various vitreoretinal indications in the paediatric population. METHODS: Consecutive patients aged 18 years or less undergoing vitrectomy for various vitreoretinal indications over a 5-year period were studied retrospectively. Two different surgical techniques were used: a modified 25-gauge approach in which the sclerotomies and conjunctiva were sutured as described previously for most children under the age of 1 year, and a transconjunctival 25-gauge approach for older children RESULTS: 56 eyes in 49 children (16 girls and 33 boys) were included. Intraoperative unplanned events or complications included: conversion to 20-gauge vitrectomy (four), conversion of one port to a 20-gauge sclerotomy (two), suspected lens damage (one) and intraoperative bleeding from a vascular ridge (one). Postoperative complications included cataract (five), rhegmatogenous retinal detachment (four) and vitreous haemorrhage (three). The four retinal detachments were either recurrent or occurred in eyes with complex ocular pathology and were not felt to be related to the surgical technique. There were no cases of postoperative hypotony requiring intervention, choroidal detachment, endophthalmitis or sclerotomy-related retinal breaks. CONCLUSIONS: 25-gauge vitreoretinal techniques can be used in various paediatric vitreoretinal conditions and facilitate easy access to small spaces in the paediatric eye. To avoid postoperative hypotony, a modified technique is recommended for younger babies in which the conjunctiva and sclera is sutured.

Combined 25- and 23-gauge surgery: a new sutureless vitrectomy technique.

The 25- and 23-gauge sutureless vitrectomy techniques decrease the surgical trauma and improve patients' postoperative comfort. The disadvantages of the 25-gauge system include pliable instrument and slower gel removal. The 23-gauge system provides faster speed of vitrectomy and the instruments have stiffer shafts, but it requires a larger incision. To circumvent some of these limitations, a new sutureless transconjunctival vitrectomy technique that combines 23- and 25-gauge cannulas and instruments was studied. Fifty-three patients underwent vitreoretinal surgery using two 25-gauge ports for the infusion and light probe and one 23-gauge port for the vitreous cutter and various instruments. No intraoperative or postoperative complications were noted and no cases required conversion to another technique.

Protein dynamics and catalysis: the problems of transition state theory and the subtlety of dynamic control.

This manuscript describes ongoing research on the nature of chemical reactions in enzymes. We will investigate how protein dynamics can couple to chemical reaction in an enzyme. We first investigate in some detail why transition state theory cannot fully describe the dynamics of chemical reactions catalysed by enzymes. We describe quantum theories of chemical reaction in condensed phase including studies of how the symmetry of coupled vibrational modes differentially affects reaction dynamics. We make reference to previous work in our group on a variety of condensed phase chemical reactions (liquid and crystalline) and a variety of enzymatically catalysed reactions including the reactions of lactate dehydrogenase and purine nucleoside phosphorylase. All the protein motions we have studied have been quite rapid. We will propose methods to find motions over a broad range of time-scales in enzymes that couple to chemical catalysis. We report recent findings which show that conformational fluctuations in lactate dehydrogenase can strongly affect its ability to catalyse reactions through protein motion, and that only a tiny minority of conformations appear to be catalytically competent.

The fellow eye of patients with phakic rhegmatogenous retinal detachment from atrophic holes of lattice degeneration without posterior vitreous detachment.

BACKGROUND: Primary phakic rhegmatogenous retinal detachment (RRD) without posterior vitreous detachment (PVD) represents a unique clinical entity that behaves differently from RRD associated with PVD. While previous studies have reported the long term findings in the fellow eye of patients with RRD and PVD, the outcome of the fellow eye of patients with RRD without PVD is not known. METHODS: Consecutive patients with RRD not associated with PVD were studied retrospectively. The authors evaluated the fellow eye for retinal detachment or other vision threatening pathology. RESULTS: 27 patients (mean age 32 years) were studied with follow up of between 9 and 326 months (mean 111 months). 24 (89%) were myopic. Bilateral retinal detachment occurred in eight patients (30%). On initial examination, 17 patients (63%) had retinal findings (including lattice degeneration, atrophic holes, and/or cystic retinal tufts) in the fellow eye that might predispose them to retinal detachment. 14 vision threatening events or diagnoses occurred (nine of which were rhegmatogenous in nature) in the fellow eye including eight retinal detachments, one traumatic PVD without retinal tears, one retinal tear after PVD, one diagnosis of pigmentary glaucoma needing trabeculectomy, two visually significant cataracts, and one diagnosis of chorioretinitis. 23 patients (85%) maintained visual acuity better than 20/50, with most retaining 20/20 vision in the fellow eye. CONCLUSIONS: Patients who experience RRD without PVD are at risk of developing vision threatening events in the contralateral eye and, as such, the fellow eye should be followed carefully.

Horner's syndrome and dissection of the internal carotid artery after chiropractic manipulation of the neck.

PURPOSE: To report a case of Horner's syndrome and dissection of the internal carotid artery after chiropractic manipulation of the neck. METHODS: Case report. A 44-year-old woman with no prior ocular or vascular history presented with severe right-sided head and neck pain, ptosis, and miosis following chiropractic treatment for a strained right shoulder muscle. RESULTS: Magnetic resonance angiography of the neck and brain revealed a dissection of the right internal carotid artery as well as a suggestion of subtle dissection in the right vertebral artery. No significant brain abnormalities were noted on magnetic resonance imaging. Pharmacological testing was consistent with preganglionic oculosympathetic damage. CONCLUSION: Acute, painful Horner's syndrome as a manifestation of vascular dissection may be associated with chiropractic manipulation of the neck.

Indocyanine green angiography of the anterior segment in patients undergoing strabismus surgery.

BACKGROUND: Anterior segment imaging using fluorescein angiography is only suitable in lightly pigmented irides as the brown pigmentation of the iris masks fluorescein transmission. Indocyanine green (ICG) angiography has excellent penetration of pigment epithelium and, therefore, has potential application in detecting perfusion changes of dark irides after strabismus surgery. METHODS: A prospective study was conducted on patients older than 15 years undergoing strabismus surgery. A fundus camera was focused on the arteriolar tufts of the pupillary margin and 50 mg of ICG (concentration of 12.5 mg/ml) was given intravenously. Images were then obtained at 1 minute intervals of 5 minutes' duration. RESULTS: 45 patients with a mean age of 54.6 years and a mean follow up period of 8.6 weeks were studied. There were 23 patients in the primary surgery group, 11 in the secondary surgery group, and 11 in the staged group. Iris ICG angiograms were successfully performed in all patients. No persistent filling defect was detected in the primary and secondary horizontal recti surgery groups or in the secondary or staged vertical and combined vertical rectus groups 6-8 weeks postoperatively. 57% of both primary vertical and combined vertical and horizontal groups showed defects in the early postoperative phase. Only three cases demonstrated late perfusion defects in this series. CONCLUSION: ICG can detect iris perfusion changes in dark irides after strabismus surgery. Iris reperfusion was achieved in the majority of the cases.

Neural network methods for identification and optimization of quantum mechanical features needed for bioactivity.

This paper presents a new approach to the discovery and design of bioactive compounds. The focus of this application will be on the analysis of enzymatic inhibitors. At present the discovery of enzymatic inhibitors for therapeutic use is often accomplished through random searches. The first phase of discovery is a random search through a large pre-fabricated chemical library. Many molecules are tested with refined enzyme for signs of inhibition. Once a group of lead compounds have been discovered the chemical intuition of biochemists is used to find structurally related compounds that are more effective. This step requires new molecules to be conceived and synthesized, and it is the most time-consuming and expensive step. The development of computational and theoretical methods for prediction of the molecular structure that would bind most tightly prior to synthesis and testing, would facilitate the design of novel inhibitors. In the past, our work has focused on solving the problem of predicting the bioactivity of a molecule prior to synthesis. We used a neural network trained with the bioactivity of known compounds to predict the bioactivity of unknown compounds. In our current work, we use a separate neural network in conjunction with a trained neural network in an attempt to gain insight as to how to modify existing compounds and increase their bioactivity.

Telemedical evaluation and management of retinopathy of prematurity using a fiberoptic digital fundus camera.

OBJECTIVE: We sought to determine whether retinopathy of prematurity (ROP) can be evaluated and managed telemedically. DESIGN: Multicenter noncomparative case series. PARTICIPANTS: Ten patients (19 eyes) with ROP were evaluated and treated per standard of care and imaged with the RetCam 120 digital fundus camera (Massie Research Laboratories, Inc., Dublin, CA). INTERVENTION: Images were transmitted to a remote site for evaluation and management recommendations. MAIN OUTCOME MEASURES: Telemedical evaluations and management recommendations were compared with traditional on-site standard of care evaluations and treatments. RESULTS: The identification of Plus disease at the remote site was accurately identified in 95% of eyes. Prethreshold, threshold, and stage 4 or 5 ROP were correctly detected in 17 of 19 (89%) eyes. CONCLUSIONS: Results indicate ROP can be evaluated and treatment recommendations made at remote sites with telemedicine strategies.

Prediction of inhibitor binding free energies by quantum neural networks. Nucleoside analogues binding to trypanosomal nucleoside hydrolase.

A computational method has been developed to predict inhibitor binding energy for untested inhibitor molecules. A neural network is trained from the electrostatic potential surfaces of known inhibitors and their binding energies. The algorithm is then able to predict, with high accuracy, the binding energy of unknown inhibitors. IU-nucleoside hydrolase from Crithidia fasciculata and the inhibitor molecules described previously [Miles, R. W. Tyler, P. C. Evans, G. Furneaux R. H., Parkin, D. W., and Schramm, V. L. (1999) Biochemistry 38, xxxx-xxxx] are used as the test system. Discrete points on the molecular electrostatic potential surface of inhibitor molecules are input to neural networks to identify the quantum mechanical features that contribute to binding. Feed-forward neural networks with back-propagation of error are trained to recognize the quantum mechanical electrostatic potential and geometry at the entire van der Waals surface of a group of training molecules and to predict the strength of interactions between the enzyme and novel inhibitors. The binding energies of unknown inhibitors were predicted, followed by experimental determination of K(i)() values. Predictions of K(i)() values using this theory are compared to other methods and are more robust in estimating inhibitory strength. The average deviation in estimating K(i)() values for 18 unknown inhibitor molecules, with 21 training molecules, is a factor of 5 x K(i)() over a range of 660 000 in K(i)() values for all molecules. The a posteriori accuracy of the predictions suggests the method will be effective as a guide for experimental inhibitor design.

Estrogen receptor in the human eye: influence of gender and age on gene expression.

PURPOSE: Many epidemiologic studies indicate an increased incidence of certain vision threatening conditions in postmenopausal women. These data suggest that changes in sex steroid homeostasis may affect the physiology of the eye. To provide support to this hypothesis, the expression of estrogen receptor alpha (ERalpha) in human eye tissues was investigated. METHODS: Complementary studies including RNA analysis by reverse transcription polymerase chain reaction, western blot analysis, and immunocytochemistry were used to provide evidence of ERalpha expression. Protein detection was carried out using a mouse monoclonal antibody raised against an epitope located in the ligand binding domain of the human receptor. Cellular localization was studied on formalin-fixed paraffin-embedded eye sections using conventional immunohistochemical techniques. RESULTS: Gender and age differences in ERalpha mRNA expression were observed in retina. The 65-kDa ERalpha protein was detected in the retina and retinal pigment epithelium (RPE) of young female eyes but not in eye tissues dissected from men and postmenopausal women. Immunocytochemistry corroborated ERalpha staining of a young female neurosensory retina and RPE. In addition, ERalpha could be detected in the ciliary body, in the iris, and in the epithelium of the lens. CONCLUSIONS: The presence of the ERalpha in the human eye suggests that the sex steroid hormone axis may play a role in the pathogenesis of certain ocular diseases.

Treatment of pigment epithelial detachments due to age-related macular degeneration with intra-ocular C3F8 injection.

PURPOSE: To flatten pigment epithelial detachments (PED) due to age-related macular degeneration in an attempt to visualize the underlying choroidal neovascularization by fluorescein angiography (FA) and reveal a treatment target. METHODS: Nine patients with PED received intravitreal gas injections via the pars plana and postured face down. Fluorescein angiograms were obtained before and after gas injection. In two patients, retinal scotopic sensitivity was also measured. RESULTS: Eight patients demonstrated change in the shape and size of the PED following gas injection. Four patients showed a better delineation of underlying structures on FA. Three patients had focal laser treatment to the newly visible choroidal neovascular complex, but this was successful in only one patient with flattening of the PED. CONCLUSION: Pigment epithelial detachments can be modified by intravitreal gas injection in some patients, but this treatment did not have a major impact on overall outcome or management.

Systemic risk factors for idiopathic macular holes: a case-control study.

PURPOSE/BACKGROUND: The idiopathic full-thickness macular hole (IFTMH) is an important cause of poor vision in the elderly affecting predominantly women over the age of 60 years. While it is accepted that vitreoretinal traction is an important local factor in the development of IFTMH, the underlying cause is not known. The aim of this study was to identify possible systemic risk factors for the development of IFTMH. METHODS: Two hundred and thirty-seven patients with IFTMH (cases) attending the Macular Hole Clinic at Moorfields Eye Hospital were identified. These were compared with 172 patients without macular holes (controls) attending other clinics in the same hospital. Cases and controls were frequency-matched by sex. The prevalence of the following factors in both groups was ascertained by interview: ethnic origin, place of birth, housing tenure, any systemic diseases, current and lifetime consumption of medication, severe dehydrational episodes, menstrual and obstetric history, onset and severity of menopause and use of exogenous oestrogens (in women only), osteoporosis, vegetarianism, use of vitamin supplementation, and smoking and alcohol consumption. Height and weight were measured for all participants. RESULTS: Cases of IFTMH macular holes were predominantly women (67%) and aged 65 years and older (74%). We found very few systemic risk factors that were significantly associated with IFTMH. There was a higher prevalence of diabetes in controls (12% vs 5%). There was no association between the majority of indicators of oestrogen exposure in women and macular holes, but cases had a more difficult menopause as judged by the severity of hot flushes at menopause: odds ratio 2.6 (1.4-4.6). CONCLUSIONS: In common with other studies, we found only a few systemic factors associated with IFTMH. The study did confirm, however, that IFTMH is a strongly gender-related disease. There is some evidence for the role of sudden changes in hormonal balance, as seen by the increased reporting of severity of symptoms around the menopause along with (statistically non-significant) increased risks associated with hysterectomy and oophorectomy. The particular aetiological factor which puts women at increased risk of macular holes requires further studies.

The use of corticosteroids for choroidal neovascularisation in young patients.

PURPOSE: To investigate the role of systemic corticosteroids in the treatment of sight-threatening choroidal neovascularisation (CNV) in patients with punctate inner choroidopathy (PIC) and multifocal inner choroiditis (MIC). METHODS: Twelve eyes of 10 patients with evidence of PIC or MIC with recent visual symptoms were identified. All eyes had CNV within the foveal avascular zone on fundus fluorescein angiography (FFA). Systemic oral prednisolone at an initial dose of 1 mg/kg (60-80 mg) was given for 3-5 days and the dose was subsequently tapered. Changes in best corrected visual acuity and leakage on FFA were recorded during follow-up. Systemic side-effects of the corticosteroids were monitored. RESULTS: In 10 of 12 eyes vision improved or stabilised. Leakage on FFA resolved in 9 eyes and was reduced in 3. Four patients required more than one course of oral corticosteroids. One patient was maintained on low-dose oral corticosteroids for recurrent CNV activity. No systemic complications from the treatment were observed. CONCLUSION: A course of oral corticosteroids in healthy young patients with subfoveal CNV in PIC or MIC may reduce subretinal vascular leakage and stabilise vision when no other proven treatment option is available.

Proliferative vitreoretinopathy: a natural history of the fellow eye.

OBJECTIVE: To determine how often the fellow eyes of patients with proliferative vitreoretinopathy (PVR) harbor a vision-threatening condition at presentation; to determine how often the fellow eyes of patients with PVR develop vision-threatening conditions; and to determine how often the fellow eyes of patients with PVR lose vision. DESIGN: A retrospective case review design was used. PARTICIPANTS: Two hundred and forty-nine patients with PVR were studied. INTERVENTION: The authors observed the fellow eye of eyes with PVR for vision-threatening pathology. MAIN OUTCOME MEASURES: The primary anatomic endpoint of this study was the detection of vision-threatening pathology in the fellow eye of patients with PVR. Secondary outcome measures included the development of visual loss in the fellow eye. RESULTS: A wide variety of vision-threatening conditions were diagnosed in the fellow eyes of patients with PVR. Of patients meeting entry criteria with reliable follow-up data, greater than 50% of fellow eyes demonstrated vision-threatening pathology at some point during follow-up. CONCLUSIONS: Patients who develop PVR in one eye are at considerable risk for developing vision-threatening pathology or vision-damaging conditions in the fellow eye. This information should be carefully considered when making surgical decisions in patients facing PVR surgery.

Large kinetic isotope effects in enzymatic proton transfer and the role of substrate oscillations.

We propose an interpretation of the experimental findings of Klinman and coworkers [Cha, Y., Murray, C. J. & Klinman, J. P. (1989) Science 243, 1325-1330; Grant, K. L. & Klinman, J. P. (1989) Biochemistry 28, 6597-6605; and Bahnson, B. J. & Klinman, J. P. (1995) Methods Enzymol. 249, 373-397], who showed that proton transfer reactions that are catalyzed by bovine serum amine oxidase proceed through tunneling. We show that two different tunneling models are consistent with the experiments. In the first model, the proton tunnels from the ground state. The temperature dependence of the kinetic isotope effect is caused by a thermally excited substrate mode that modulates the barrier, as has been suggested by Borgis and Hynes [Borgis, D. & Hynes, J. T. (1991) J. Chem. Phys. 94, 3619-3628]. In the second model, there is both over-the-barrier transfer and tunneling from excited states. Finally, we propose two experiments that can distinguish between the possible mechanisms.