Limbal stem cell deficiency secondary to systemic paclitaxel (Taxol) for breast cancer: a case report.

BACKGROUND: Paclitaxel (PTX) is an antineoplastic drug widely used in treatments for ovarian, breast, and small-cell lung cancer. Although ocular effects associated with PTX have been previously described, very few studies have specifically reported systemic PTX as a contributing factor for limbal stem cell deficiency (LSCD), which is characterized by the loss of stem cell and barrier function of the limbus leading to progressive pain and reduction in visual acuity. Described here is a unique case where a patient was diagnosed with LSCD secondary to PTX use for the treatment of breast cancer, at doses of PTX far lower than what is reported in current literature. CASE PRESENTATION: A 73-year-old woman with a previous diagnosis of breast cancer with liver metastasis presented with a complaint of increasing pain in the left eye more than the right, along with decreasing visual acuity in both eyes following 3 months of PTX therapy for recurrent liver metastases. Upon examination, best-corrected visual acuity was 20/100 in the right eye and counting fingers on the left. Peripheral neovascularization, stromal scarring, and features of limbal stem cell deficiency (LSCD) were noted on the right cornea. A central neurotrophic ulcer with thinning to 50% and 360 degrees of conjunctivalization were noted on the left. After the discontinuation PTX with doxorubicin as the substitute, there was no further progression of her LSCD, and stabilization of her ocular surface was achieved. CONCLUSION: Although chemotherapy induced LSCD is a relatively rare adverse event, it is essential for clinicians starting new chemotherapy agents to consider the potential ocular toxicities that may result in their use. Ophthalmology review is recommended for patients after starting PTX therapy to assess for signs of LSCD, particularly in patients where drug toxicity can be aggravated due to impaired hepatic function.

Topography-guided laser refractive surgery.

PURPOSE OF REVIEW: Topography-guided laser refractive surgery regularizes the front corneal surface irregularities to achieve the desired refractive outcome. This is particularly applicable in highly aberrated corneas, where wavefront aberrometry is often not possible. This article aims to review the recently published results of topography-guided ablations in normal regular corneas, highly aberrated corneas, and its application in conjunction with collagen cross-linking (CXL) in cases of keratectasia. RECENT FINDINGS: Topography-guided laser ablation is increasingly used with good efficacy and safety outcomes in highly aberrated corneas with irregular astigmatism. These include eyes with refractive surgery complications including postlaser in-situ keratomileusis ectasia, decentered ablation, small optical zones, asymmetrical astigmatism, and postradial keratectomy astigmatism. Further indications are for postkeratoplasty astigmatism and keratoconus. Simultaneous topography-guided ablations with CXL in keratectasia have been promising, both in addressing the surface irregularities and progressive nature of the conditions. SUMMARY: Topography-guided laser refractive surgery is proving to be effective and well tolerated in the visual rehabilitation of highly aberrated eyes, with increasing predictability based on the recent research.

Clinical results of topography-based customized ablations in highly aberrated eyes and keratoconus/ectasia with cross-linking.

PURPOSE: To report results of a series of highly aberrated corneas treated with a topography-guided excimer laser ablation. METHODS: Retrospective, nonrandomized, consecutive series of eyes treated with topography-guided photorefractive keratectomy (TG-PRK) with the customized topographical neutralization technique (TNT). Cases included postoperative refractive surgery decentered ablations, optical zone enlargement, asymmetrical astigmatism, postoperative radial keratotomy (RK), postoperative keratoplasty, keratoconus combined with collagen cross-linking (CXL), and postoperative LASIK ectasia combined with CXL. Uncorrected distance visual acuity (UDVA), corrected distance visual acuity (CDVA), and manifest refraction were analyzed preoperatively and 6 months postoperatively. RESULTS: In decentered ablation cases, 94% of 37 eyes were within 1.00 diopter (D) of the attempted refractive outcome, with 76% within 0.50 D. Mean topographic, central, optical zone of uniform (monodioptric) power increased from 3.5 to 5.2 mm in 25 eyes. Thirty-one eyes treated for asymmetrical astigmatism showed improvement in cylinder from mean 1.31 to 0.52 D. Ten of 11 eyes treated for previous RK astigmatism achieved postoperative UDVA 20/40 or better. Twenty-seven eyes with postoperative keratoplasty astigmatism were treated, with 7 (25.9%) eyes gaining > or = 2 lines and 12 (44.4%) eyes gaining > or = 1 line of CDVA. Of eyes with keratoconus that were treated using TG-PRK with CXL, 42 (58%) eyes had UDVA 20/40 or better, and 66 (92%) eyes had CDVA 20/40 or better. Twelve (71%) of 17 eyes treated for postoperative LASIK ectasia using TG-PRK with CXL had UDVA 20/40 or better. Nine (53%) eyes gained > or = 2 lines of CDVA. CONCLUSIONS: Topography-guided laser treatment with custom TNT, combined with CXL in keratoconus and ectasia, is an effective, safe, and increasingly predictable option for highly aberrated corneas.

Larval settlement preference maximizes genetic mixing in an inbreeding population of a simultaneous hermaphrodite (Bugula stolonifera, Bryozoa).

Conspecific aggregations in terrestrial and aquatic organisms can have a significant effect on an individual's survival, growth and reproductive fitness, particularly if these aggregations are composed of closely related individuals. Such aggregations can form passively, as a consequence of dispersal, or actively, as a consequence of kin recognition. In this study, we investigated the genetic composition of individuals in conspecific aggregations in the simultaneous hermaphroditic marine bryozoan Bugula stolonifera. Conspecific larvae routinely metamorphose on adult colonies; the possibility that larvae select or avoid their maternal colony was investigated utilizing 10 newly developed polymorphic microsatellite loci. Adult colonies were collected from Eel Pond, Woods Hole, Massachusetts and inspected for the presence of attached individuals. Adult colonies and their attached individuals were genotyped and compared to assess genetic relatedness within and among these groups relative to the overall genetic variability of the sampling site. Overall, the population of B. stolonifera at this site was found to be outside Hardy-Weinberg equilibrium because of significant levels of inbreeding. No significant genetic differentiation, however, was found between any groups, documenting that a group containing an adult colony and its attached individuals had as much genetic variability as was found for the entire sampling site. Parentage-exclusion analyses showed that the vast majority of attached individuals (>93%) could not have derived from the colony on which they were attached. Kinship analyses showed that the majority of attached individuals (≈63%) shared less than a half-sibling relationship. These results suggest that a colony's nearest neighbours are not composed of siblings, and thus, larval settlement preference can maximize outcrossing in this inbreeding population.

Integrated HPLC-MS and (1)H-NMR spectroscopic studies on acyl migration reaction kinetics of model drug ester glucuronides.

Acyl glucuronides (AGs) are common, chemically reactive metabolites of acidic xenobiotics. Concerns about the potential of this class of conjugate to cause toxicity in man require efficient methods for the determination of reactivity, and this is commonly done by measuring transacylation kinetics. High-performance liquid chromatography-mass spectrometry (HPLC-MS) and nuclear magnetic resonance (NMR) spectroscopy were applied to the kinetic analysis of AG isomerization and hydrolysis for the 1-beta-O-AGs of ibufenac, (R)- and (S)-ibuprofen, and an alpha,alpha-dimethylated ibuprofen analogue. Each AG was incubated in either aqueous buffer at pH 7.4 or human plasma at 37 degrees C. Aliquots of these samples, taken throughout the reaction time course, were analysed by HPLC-MS and (1)H-NMR spectroscopy and the results compared. For identification of the AGs incubated in pH 7.4 buffer and for analysis of kinetic rates, (1)H-NMR spectroscopy generally gave the most complete set of data, but for human plasma the use of (1)H-NMR spectroscopy was impractical and HPLC-MS was more suitable. HPLC-MS was more sensitive than (1)H-NMR spectroscopy, but the lack of suitable stable-isotope labelled internal standards, together with differences in response between glucuronides and aglycones, made quantification problematic. Using HPLC-MS a specific 1-beta-O-AG-related ion at m/z 193 (the glucuronate fragment) was noted enabling selective determination of these isomers. In buffer, transacylation reactions predominated, with relatively little hydrolysis to the free aglycone observed. In human plasma incubations the observed rates of reaction were much faster than for buffer, and hydrolysis to the free aglycone was the major route. These results illustrate the strengths and weaknesses of each analytical approach for this class of analyte.

Is the cell division cycle gated by a circadian clock? The case of Chlamydomonas reinhardtii.

Circadian oscillators are known to regulate the timing of cell division in many organisms. In the case of Chlamydomonas reinhardtii, however, this conclusion has been challenged by several investigators. We have reexamined this issue and find that the division behavior of Chlamydomonas meets all the criteria for circadian rhythmicity: persistence of a cell division rhythm (a) with a period of approximately 24 h under free-running conditions, (b) that is temperature compensated, and (c) which can entrain to light/dark signals. In addition, a mutation that lengthens the circadian period of the phototactic rhythm similarly affects the cell division rhythm. We conclude that a circadian mechanism determines the timing of cell division in Chlamydomonas reinhardtii.

Intracellular pH of sea urchin eggs measured by the dimethyloxazolidinedione (DMO) method.

Intracellular pH (pH1) of sea urchin eggs and embryos was determined using DMO (5,5-dimethyl-2,4-oxazolidinedione). By this method, the pH1 of Lytechinus pictus eggs increased after fertilization from 6.86 to 7.27, and this higher pHi was maintained thereafter, as has been previously observed with pH microelectrodes. The same general result was obtained with the eggs of Strongylocentrotus purpuratus, in contrast to previous estimates of the pH of egg homogenates from this species, which had indicated a rise and then fall of pHi after fertilization. pHi did not significantly change during early cell divisions. Studies of treatments that alter pHi confirmed that ammonia alkalizes and acetate acidifies the cells. The regulation of pHi by embryos in the acidic seawater is impaired if sodium is absent, whereas unfertilized eggs can regulate pHi in acidic, sodium-free seawater.

Compartmentalization of algal bioluminescence: autofluorescence of bioluminescent particles in the dinoflagellate Gonyaulax as studied with image-intensified video microscopy and flow cytometry.

Compartmentalization of specialized functions to discrete locales is a fundamental theme of eucaryotic organization in cells. We report here that bioluminescence of the dinoflagellate alga Gonyaulax originates in vivo from discrete subcellular loci that are intrinsically fluorescent. We demonstrate this localization by comparing the loci of fluorescence and bioluminescence as visualized by image-intensified video microscopy. These fluorescent particles appeared to be the same as the previously described in vitro "scintillons." We attribute the endogenous fluorescence to that of the bioluminescence substrate, luciferin, because (a) the fluorescence excitation and emission characteristics are comparable, (b) the autofluorescence is lost after exhaustive stimulation of bioluminescence, and (c) the fluorescence of discharged particles in vitro can be restored by adding luciferin. The fluorescence in vivo exhibits a standard property of circadian (daily) rhythmicity: under constant environmental conditions, the intensity of the particle fluorescence fluctuates cyclically (it is maximal during the night phase and is low during the day). Thus, luciferin is localized within the cell at discrete loci from which the bioluminescence emanates; the cellular quantity of luciferin is rhythmically modulated by the circadian clock.

Characterization of the bioluminescent organelles in Gonyaulax polyedra (dinoflagellates) after fast-freeze fixation and antiluciferase immunogold staining.

To characterize the microsources of bioluminescent activity in the dinoflagellate Gonyaulax polyedra, an immunogold labeling method using a polyclonal antiluciferase was combined with fast-freeze fixation and freeze substitution. The quality of the preservation and the specificity of the labeling were greatly improved compared to earlier results with chemical fixation. Two organelles were specifically labeled: cytoplasmic dense bodies with a finely vermiculate texture, and mature trichocysts, labeled in the space between the shaft and the membrane. The available evidence indicates that the dense bodies are the light-emitting microsources observed in vivo. The dense bodies appear to originate in the Golgi area as cytoplasmic densifications and, while migrating peripherally, come into contact with the vacuolar membrane. Mature organelles protrude and hang like drops in the vacuolar space, linked by narrow necks to the cytoplasm. These structural relationships, not previously apparent with glutaraldehyde fixation, suggest how bioluminescent flashes can be elicited by a proton influx from a triggering action potential propagated along the vacuolar membrane. Similar dense bodies were labeled in the active particulate biochemical fraction (the scintillons), where they were completely membrane bound, as expected if their necks were broken and resealed during extraction. The significance of the trichocyst reactivity remains enigmatic. Both organelles were labeled with affinity-purified antibody, which makes it unlikely that the trichocyst labeling is due to a second antibody of different specificity. But trichocysts are not bioluminescent; the cross-reacting material could be luciferase present in this compartment for some other reason, or a different protein carrying similar antigenic epitopes.

Circadian changes in enzyme concentration account for rhythm of enzyme activity in gonyaulax.

A circadian rhythm in the activity of luciferase is partly responsible for rhythmic bioluminescence in the dinoflagellate alga Gonyaulax polyedra. The cyclic activity of this enzyme can be attributed to a corresponding rhythm in the concentration of immunologically reactive luciferase protein. Hence protein turnover (synthesis or degradation or both) is used by the endogenous clock to control the daily rhythm of bioluminescence.

Circadian oscillations of cytosolic and chloroplastic free calcium in plants.

Tobacco and Arabidopsis plants, expressing a transgene for the calcium-sensitive luminescent protein apoaequorin, revealed circadian oscillations in free cytosolic calcium that can be phase-shifted by light-dark signals. When apoaequorin was targeted to the chloroplast, circadian chloroplast calcium rhythms were likewise observed after transfer of the seedlings to constant darkness. Circadian oscillations in free calcium concentrations can be expected to control many calcium-dependent enzymes and processes accounting for circadian outputs. Regulation of calcium flux is therefore fundamental to the organization of circadian systems.

Circadian clock mutants of cyanobacteria.

A diverse set of circadian clock mutants was isolated in a cyanobacterial strain that carries a bacterial luciferase reporter gene attached to a clock-controlled promoter. Among 150,000 clones of chemically mutagenized bioluminescent cells, 12 mutants were isolated that exhibit a broad spectrum of periods (between 16 and 60 hours), and 5 mutants were found that show a variety of unusual patterns, including arrhythmia. These mutations appear to be clock-specific. Moreover, it was demonstrated that in this cyanobacterium it is possible to clone mutant genes by complementation, which provides a means to genetically dissect the circadian mechanism.

Expression of a gene cluster kaiABC as a circadian feedback process in cyanobacteria.

Cyanobacteria are the simplest organisms known to have a circadian clock. A circadian clock gene cluster kaiABC was cloned from the cyanobacterium Synechococcus. Nineteen clock mutations were mapped to the three kai genes. Promoter activities upstream of the kaiA and kaiB genes showed circadian rhythms of expression, and both kaiA and kaiBC messenger RNAs displayed circadian cycling. Inactivation of any single kai gene abolished these rhythms and reduced kaiBC-promoter activity. Continuous kaiC overexpression repressed the kaiBC promoter, whereas kaiA overexpression enhanced it. Temporal kaiC overexpression reset the phase of the rhythms. Thus, a negative feedback control of kaiC expression by KaiC generates a circadian oscillation in cyanobacteria, and KaiA sustains the oscillation by enhancing kaiC expression.

Light-emitting diode flashlights as effective and inexpensive light sources for fluorescence microscopy.

Light-emitting diodes (LEDs) are becoming more commonly used as light sources for fluorescence microscopy. We describe the adaptation of a commercially available light-emitting diode flashlight for use as a source for fluorescence excitation. This light source is long-lived, inexpensive and is effective for excitation in the range of 440-600 nm.

The cyanobacterial circadian system: a clock apart.

Several new molecular components of the circadian clocks of animals, fungi, and bacteria have been unveiled in the past two years. Enough parts are now identified to indicate that there is more than one way to build a biological clock, although there are parallels in the cycling molecular events among disparate groups of organisms.

Molecular analyses of circadian gene variants reveal sex-dependent links between depression and clocks.

An extensive literature links circadian irregularities and/or sleep abnormalities to mood disorders. Despite the strong genetic component underlying many mood disorders, however, previous genetic associations between circadian clock gene variants and major depressive disorder (MDD) have been weak. We applied a combined molecular/functional and genetic association approach to circadian gene polymorphisms in sex-stratified populations of control subjects and case subjects suffering from MDD. This approach identified significant sex-dependent associations of common variants of the circadian clock genes hClock, hPer3 and hNpas2 with major depression and demonstrated functional effects of these polymorphisms on the expression or activity of the hCLOCK and hPER3 proteins, respectively. In addition, hCLOCK expression is affected by glucocorticoids, consistent with the sex-dependency of the genetic associations and the modulation of glucocorticoid-mediated stress response, providing a mechanism by which the circadian clock controls outputs that may affect psychiatric disorders. We conclude that genetic polymorphisms in circadian genes (especially hClock and hPer3, where functional assays could be tested) influence risk of developing depression in a sex- and stress-dependent manner. These studies support a genetic connection between circadian disruption and mood disorders, and confirm a key connection between circadian gene variation and major depression.

Adaptive significance of circadian programs in cyanobacteria.

Prokaryotic cyanobacteria express robust circadian (daily) rhythms, even when growing with doubling times that are considerably faster than once every 24 h. This biological clock orchestrates cellular events to occur in an optimal temporal program. Competition experiments demonstrate that fitness is enhanced when the circadian period resonates with the period of the environmental cycle.

Light pulses induce "singular" behavior and shorten the period of the circadian phototaxis rhythm in the CW15 strain of Chlamydomonas.

While measuring action spectra for phase-shifting the circadian clock of Chlamydomonas, we observed that light pulses started near the phase response curve (PRC) "breakpoint" caused a reduction of the amplitude of the phototactic rhythm and two unexpected effects: (1) nonmonotonic fluence response curves (FRCs), and (2) shortening of the period of the subsequent free-running rhythm. The reduction of the rhythm's amplitude is dependent upon both the fluence and wavelength of the light pulse. The results are consistent with the amplitude being dependent upon the perceived "strength" of the stimulus, and with the nonmonotonic FRCs and reduced amplitude reflecting a light-induced change of the pacemaker's state variables to a region of the phase plane close to the "singularity." The period change that is evoked by single stimuli exhibits novel characteristics: large changes in period and a phase specificity that correlates with "singular" behavior. These period changes also appear to be a function of the stimulus strength, but indirectly; the magnitude of the period change is most strongly correlated with the magnitude of the light-induced phase shift. These results are interpreted in the context of limit cycle models of circadian clocks, and are used to suggest new tactics for measuring action spectra of light-induced clock resetting.

Cycloheximide inhibits light-induced phase shifting of the circadian clock in Neurospora.

Cycloheximide inhibits light-induced phase shifting of the circadian clock and protein synthesis in Neurospora. Light resetting is not inhibited in mutants whose protein synthesis is resistant to cycloheximide. When light and cycloheximide are presented together at various circadian phases, the final phase shift is always determined by cycloheximide. This dual-treatment phase response curve approach may be useful for other studies using pharmacological treatments to analyze clock pathways. Taken together, the results suggest that synthesis of a protein (or proteins) is involved in the phototransduction pathway of the circadian clock in Neurospora.

Circadian phototransduction: phase resetting and frequency of the circadian clock of Gonyaulax cells in red light.

Constant red light (RR) influences the Gonyaulax clock in several ways: (1) Phase resetting by white or blue light pulses is stronger under background RR than in constant white light (WW); (2) frequency of the rhythm is less in RR than in WW; and (3) the amplitude of the spontaneous flashing rhythm is greater in RR than in WW. The phase response curve (PRC) to 4-hr white or blue light pulses is of high amplitude (Type 0) for cells in RR, but is of lower amplitude (Type 1) for cells in WW. In all cases, the PRC is highly asymmetrical: The magnitude of advance phase resetting is far higher than that of delay resetting. Consistent with this PRC, Gonyaulax cells in RR (free-running period greater than 24 hr) will entrain to T cycles of between 21 and 26.5 hr. The bioluminescence rhythms exhibit "masking" by blue light pulses while entrained to these T cycles. The fluence response of phase resetting to light-pulse intensity is not linear or logarithmic--rather, it is discontinuous. This feature is consistent with a limit cycle interpretation of Type 0 resetting of circadian clocks. Light pulses that cause large phase shifts also shorten the subsequent free-running period. The phase angle difference between the clock and the previous LD cycle is within 2 hr of the same phase between 16 degrees C and 25 degrees C, as determined from the light PRCs at various temperatures. Several drugs that inhibit mitochondria and/or electron transport will partially inhibit the phase shift by light.