A Student-Led Methodology for Evaluating Curricular Redundancy

Background: Curricular redundancy can be a significant problem for any educational curriculum. Redundancy can be both desirable and undesirable, but differentiating the two can be quite challenging. Further, pinpointing undesirable redundancy and quantifying it so as to produce an estimate of inefficiency is even more difficult. Purpose: The purpose of this research is to describe a student-led strategy for evaluating redundancy in a highly integrated medical school curriculum. It is our hope that the methodology presented here will serve as a useful evaluation model for persons attempting similar work in various educational arenas. Setting: A highly-integrated medical school at a large public university. Intervention: This research did not require an intervention. Research Design: We identified two advanced medical students and asked them to identify redundant material across the first two years of the medical school curriculum. The students had to operationalize ‘redundancy’, develop an evaluation plan/framework, and evaluate the extent to which undesirable redundancy was prevalent in the current curriculum. Data Collection and Analysis: Students reviewed course syllabi, notes, and materials and documented the amount of redundant material they found in the curriculum. Findings: A total of approximately 167 hours, or 8.35 weeks, could be eliminated from the curriculum; the vast majority of the redundancy occurred as a result of small group activities

Mie light scattering calculations for an Indian age-related nuclear cataract with a high density of multilamellar bodies

Purpose: Multilamellar bodies (MLBs) are lipid-coated spheres (1–4 µm in diameter) found with greater frequency in the nuclear region of human age-related cataracts compared with human transparent lenses. Mie light scattering calculations have demonstrated that MLBs are potential sources of forward light scattering in human age-related nuclear cataracts due to their shape, size, frequency, and cytoplasmic contents, which often differ in refractive index from their surroundings. Previous studies have used data from several non-serial tissue sections viewed by light microscopy to extrapolate a volume and have assumed that MLBs are random in distribution. Currently, confocal microscopy is being used to examine actual tissue volumes from age-related nuclear cataracts and transparent lenses collected in India to confirm MLB shape, size, frequency, and randomness. These data allow Mie scattering calculations to be done with directly observed MLBs in intact tissue. Methods: Whole Indian donor lenses and Indian lens nuclei after extracapsular cataract extraction were immersion-fixed in 10% formalin for 24 h and in 4% paraformaldehyde for 24 h before sectioning with a Vibratome. The 160 µm thick sections were stained for 24 h in the lipid dye DiI (1,1’-dilinoleyl-3,3,3′,3′ tetramethylindocarbocyanine, 4-chlorobenzenesulfonate), washed, stabilized in Permount under coverslips and examined with a Zeiss LSM 510 confocal microscope. Individual volumes of tissue (each typically 500,000 µm3) were examined using a plan-apochromat 63X oil (NA=1.4) lens. Other lenses were prepared for electron microscopy and histological examination using previously described procedures. Results: Analysis of tissue volumes within Indian age-related nuclear cataracts and transparent lenses has confirmed that most MLBs are 1–4 µm in diameter and typically spherical with some occurring as doublets or in clusters. Most Indian cataracts and transparent lenses are similar to samples obtained in the United States. One cataract contained as many as 400,000 MLBs per mm3 –100 times more than in cataracts collected in the United States. Pairwise distribution analysis has revealed that MLBs even in this exceptional case are found with a distribution that appears to be random. Mie calculations indicate that more than 90% of the incident light could be scattered by the high density of MLBs. Conclusions: An important finding was that one advanced Indian cataract contained many more MLBs than cataracts examined from India and previously from the United States. This indicates that specific conditions or susceptibilities may exist that promote the formation of excessive MLBs. Based on the extremely high frequency, as well as their spherical shape, large size, and apparent random distribution, the MLBs are predicted according to Mie light scattering calculations to cause high amounts of forward scattering sufficient to produce nuclear opacity

Simple fixation and storage protocol for preserving the internal structure of intact human donor lenses and extracted human nuclear cataract specimens

Purpose: Increased use of phacoemulsification procedures for cataract surgeries has resulted in a dramatic decrease in the availability of cataractous nuclear specimens for basic research into the mechanism of human cataract formation. To overcome such difficulties, a fixation protocol was developed to provide good initial fixation of human donor lenses and extracted nuclei, when available, and is suitable for storing or shipping cataracts to laboratories where structural studies could be completed. Methods: Cataractous lens nuclei (n=19, ages 12 to 74 years) were obtained from operating suites after extracapsular extraction. Transparent human donor lenses (n=27, ages 22 to 92 years) were obtained from the Ramayamma International Eye Bank. After the dimensions were measured with a digital caliper, samples were preserved in 10% formalin (neutral buffered) for 24 h and followed by fixation in 4% paraformaldehyde (pH 7.2) for 48 h. Samples were stored cold (4 °C) in buffer until shipped. Samples were photographed and measured before further processing for transmission electron microscopy. Results: The dimensions of the samples varied slightly after short fixation followed by 1 to 5 months’ storage before transmission electron microscopy processing. The mean change in the axial thickness of the donor lenses was 0.15±0.21 mm or 3.0±5.4%, while that of the extracted nuclei was 0.05±0.24 mm or 1.8±7.6%. Because the initial concern was whether the nuclear core was preserved, thin sections were examined from the embryonic and fetal nuclear regions. All cellular structures were preserved, including the cytoplasm, complex edge processes, membranes, and junctions. The preservation quality was excellent and nearly equivalent to preservation of fresh lenses even for the lens cortex. Cell damage characteristic of specific nuclear cataract types was easily recognized. Conclusions: The novel fixation protocol appears effective in preserving whole donor lenses and cataractous nuclei over a wide age range. Dimensions varied only 2%–3%, and fiber cell damage correlated well with standard fixation. These methods enable researchers and clinicians in remote settings to preserve donor lenses and rare examples of extracapsular extractions for detailed examination at later times

Ultrastructural analysis of the human lens fiber cell remodeling zone and the initiation of cellular compaction

The purpose is to determine the nature of the cellular rearrangements occurring through the remodeling zone (RZ) in human donor lenses, identified previously by confocal microscopy to be about 100 µm from the capsule. Human donor lenses were fixed with 10% formalin followed by 4% paraformaldehyde prior to processing for transmission electron microscopy. Of 27 fixed lenses, ages 22, 55 and 92 years were examined in detail. Overview electron micrographs confirmed the loss of cellular organization present in the outer cortex (80 µm thick) as the cells transitioned into the RZ. The transition occurred within a few cell layers and fiber cells in the RZ completely lost their classical hexagonal cross-sectional appearance. Cell interfaces became unusually interdigitated and irregular even though the radial cell columns were retained. Gap junctions appeared to be unaffected. After the RZ (40 µm thick), the cells were still irregular but more recognizable as fiber cells with typical interdigitations and the appearance of undulating membranes. Cell thickness was irregular after the RZ with some cells compacted, while others were not, up to the zone of full compaction in the adult nucleus. Similar dramatic cellular changes were observed within the RZ for each lens regardless of age. Because the cytoskeleton controls cell shape, dramatic cellular rearrangements that occur in the RZ most likely are due to alterations in the associations of crystallins to the lens-specific cytoskeletal beaded intermediate filaments. It is also likely that cytoskeletal attachments to membranes are altered to allow undulating membranes to develop

Multilamellar spherical particles as potential sources of excessive light scattering in human age-related nuclear cataracts

The goal of this project was to determine the relative refractive index (RI) of the interior of multilamellar bodies (MLBs) compared to the adjacent cytoplasm within human nuclear fiber cells. MLBs have been characterized previously as 1-4 μm diameter spherical particles covered by multiple lipid bilayers surrounding a cytoplasmic core of variable density. Age-related nuclear cataracts have more MLBs than transparent donor lenses and were predicted to have high forward scattering according to Mie scattering theory, assuming different RIs for the MLB and cytoplasm. In this study quantitative values of relative RI were determined from specific MLBs in electron micrographs of thin sections and used to calculate new Mie scattering plots. Fresh lenses were Vibratome sectioned, immersion fixed and en bloc stained with osmium tetroxide and uranyl acetate, or uranyl acetate alone, prior to dehydration and embedding in epoxy or acrylic resins. Thin sections 70 nm thick were cut on a diamond knife and imaged without grid stains at 60 kV using a CCD camera on a transmission electron microscope (TEM). Integrated intensities in digital electron micrographs were related directly to protein density, which is linearly related to RI for a given substance. The RI of the MLB interior was calculated assuming an RI value of 1.42 for the cytoplasm from the literature. Calculated RI values for MLBs ranged from 1.35 to 1.53. Thus, some MLBs appeared to have interior protein densities similar to or less than the adjacent cytoplasm whereas others had significantly higher densities. The higher density MLBs occurred preferentially in older and more advanced cataracts suggesting a maturation process. The bilayer coats were more often observed in MLBs from transparent donors and early stage cataracts indicating that bilayer loss was part of the MLB maturation, producing large low-density spaces around dense MLB cores. These spaces were frequently observed in advanced cataracts from India as large low-density crescents and annular rings. Predicted scattering from Mie plots using particles with dense cores and low-density rims was higher than reported previously, although the most important factor was the relative RI , not the MLB coat or lack thereof. In conclusion, the measurements confirm the high protein density and RI of some MLB interiors compared to adjacent cytoplasm. This high RI ratio used in the Mie calculations suggests that for 2 000 MLBs/mm3, about half that reported for early stage nuclear cataracts from the US, the forward scattering could be more than 30% of the incident light. Therefore, the extent of forward scattering and its influence on macular visual acuity could be important components of ophthalmological evaluations of cataract patients

Medical Student Leadership Development through a Business School Partnership Model: A Case Study and Implementation Strategy

Background: There is an ongoing call for leadership development in academic health care and medical students desire more training in this area. Although many schools offer combined MD/MBA programs or leadership training in targeted areas, these programs do not often align with medical school leadership competencies and are limited in reaching a large number of students.Methods:The Leadership Initiative (LI) was a program created by a partnership between a School of Medicine (SOM) and Business School with a learning model that emphasized the progression from principles to practice, and the competencies of self-awareness, communication, and collaboration/teamwork. Through offerings across a medical school curriculum, the LI introduced leadership principles and provided an opportunity to apply them in an interactive activity or simulation. We utilized the existing SOM evaluation platform to collect data on program outcomes that included satisfaction, fidelity to the learning model, and impact.Results:From 2017 to 2020, over 70% of first-year medical students participated in LI course offerings while a smaller percentage of fourth-year students engaged in the curriculum. Most students had no prior awareness of LI course material and were equivocal about their ability to apply lessons learned to their medical school experience. Students reported that the LI offerings provided opportunities to practice the skills and competencies of self-awareness, communication, and collaboration/teamwork.Discussion: Adding new activities to an already crowded medical curriculum was the greatest logistical challenge. The LI was successful in introducing leadership principles but faced obstacles in having participants apply and practice these principles. Most students reported that the LI offerings were aligned with the foundational competencies

Ultrastructural analysis of damage to nuclear fiber cell membranes in advanced age-related cataracts from India

The primary goal was to characterize the structural alterations that occur at the fiber cell interfaces in nuclei of fully opaque cataracts removed by extracapsular cataract surgery in India. The dark yellow to brunescent nuclei, ages 38–78 years, were probably representative of advanced age-related nuclear cataracts. Thick tissue slices were fixed, en bloc stained and embedded for transmission electron microscopy. Stained thin sections contained well-preserved membranes and junctions, although the complex cellular topology often made it necessary to tilt the grid extensively to visualize the membranes. Damage to the fiber cell membranes was noted in all regions of the nucleus. The most important damage occurred within undulating membrane junctions where the loss of membrane segments was common. These membrane breaks were not sites of fusion as membrane edges were detected and cytoplasm appeared to be in contact with extracellular space, which was enlarged in many regions. Dense deposits of protein-like material were frequently observed within the extracellular space and appeared to be similar to protein in the adjacent cytoplasm. The deposits were often 20–50 nm thick, variable in length and located on specific sites on plasma membranes and between clusters of cells or cell processes. In addition, low density regions were seen within the extracellular space, especially within highly undulating membranes where spaces about 100 nm in diameter were observed. The membrane damage was more extensive and extracellular spaces were larger than in aged transparent donor lenses. Because high and low density regions contribute equally to the fluctuations in refractive index, the changes in density due to the observed damage near membranes are likely to produce significant light scattering based on theoretical analysis. The dimensions of the fluctuations in the range 20–100 nm imply that the scattering is probably similar to that of small particles that would increase high-angle scattering visible in the slit lamp. Such damage to membranes would be expected to contribute to the total opacification of the nucleus as the cataract matures. The main sources of the fluctuations appear to be the degradation of membranes and adjacent cytoplasmic proteins, as well as the redistribution of proteins and fragments

Autophagy and mitophagy participate in ocular lens organelle degradation

The eye lens consists of a layer of epithelial cells that overlay a series of differentiating fiber cells that upon maturation lose their mitochondria, nuclei and other organelles. Lens transparency relies on the metabolic function of mitochondria contained in the lens epithelial cells and in the immature fiber cells and the programmed degradation of mitochondria and other organelles occurring upon lens fiber cell maturation. Loss of lens mitochondrial function in the epithelium or failure to degrade mitochondria and other organelles in lens fiber cells results in lens cataract formation. To date, the mechanisms that govern the maintenance of mitochondria in the lens and the degradation of mitochondria during programmed lens fiber cell maturation have not been fully elucidated. Here, we demonstrate using electron microscopy and dual-label confocal imaging the presence of autophagic vesicles containing mitochondria in lens epithelial cells, immature lens fiber cells and during early stages of lens fiber cell differentiation. We also show that mitophagy is induced in primary lens epithelial cells upon serum starvation. These data provide evidence that autophagy occurs throughout the lens and that mitophagy functions in the lens to remove damaged mitochondria from the lens epithelium and to degrade mitochondria in the differentiating lens fiber cells for lens development. The results provide a novel mechanism for how mitochondria are maintained to preserve lens metabolic function and how mitochondria are degraded upon lens fiber cell maturation

Predicted Light Scattering from Particles Observed in Human Age-Related Nuclear Cataracts Using Mie Scattering Theory

PURPOSE. To employ Mie scattering theory to predict the lightscattering from micrometer-sized particles surrounded by lipid shells, called multilamellar bodies (MLBs), reported in human age-related nuclear cataracts. METHODS. Mie scattering theory is applicable to randomly distributed spherical and globular particles separated by distances much greater than the wavelength of incident light. With an assumed refractive index of 1.40 for nuclear cytoplasm, particle refractive indices from 1.33 to 1.58 were used to calculate scattering efficiencies for particle radii 0.05 to 3 m and incident light with wavelengths (in vacuo) of 400, 550, and 700 nm. RESULTS. Surface plots of scattering efficiency versus particle radius and refractive index were calculated for coated spherical particles. Pronounced peaks and valleys identified combinations of particle parameters that produce high and low scattering efficiencies. Small particles (Ͻ0.3 m radius) had low scattering efficiency over a wide range of particle refractive indices. Particles with radii 0.6 to 3 m and refractive indices 0.08 to 0.10 greater (or less) than the surrounding cytoplasm had very high scattering efficiencies. This size range corresponds well to MLBs in cataractous nuclei (average MLB radius, 1.4 m) and, at an estimated 4000 particles/mm 3 of tissue, up to 18% of the incident light was scattered primarily within a 20°forward cone. CONCLUSIONS. The calculated size of spherical particles that scatter efficiently was close to the observed dimensions of MLBs in cataractous nuclei. Particle refractive indices only 0.02 units different from the surrounding cytoplasm scatter a significant amount of light. These results suggest that the MLBs observed in human age-related nuclear cataracts may be major sources of forward light scattering that reduces contrast of fine details, particularly under dim light. (Invest Ophthalmol Vis Sci. 2007;48:303-312) DOI:10.1167/iovs.06-0480 A ssessments of light-scattering by the ocular lens depend critically on the measurement geometry. For example, slit lamp images obtained by ophthalmologists detect light scattered mainly by small particles that preferentially scatter at high angles. 1-3 Light scattered at low angles (forward scattering) is more difficult to measure in vivo, although indirect methods have been proposed, 11 Mie calculations are presented that explore parameters influencing particle-scattering efficiency and that compare predicted relative scattering from transparent and cataractous lens nuclei. It is important to note that Rayleigh and Mie scattering are not two different kinds of scattering, but are two different methods of evaluating light-scattering from particles of various sizes. Mie calculations are rigorous for spheres and correct for any size. Rayleigh calculations are correct only for very small particles, less than one tenth the wavelength of the incident light. METHODS Morphologic Analysis Data from a recent study of eight transparent and eight cataractous nuclei were used to estimate the properties of a typical MLB along the optic axis and determine the range of variables used to calculate Mie scattering surface plots. 11 Briefly, fresh lenses or nuclei were sectioned on a microtome (model 1000; Vibratome, St. Louis, MO) and the 200-m-thick sections were fixed by immersion (2% paraformaldehyde, 1% tannic acid in 0.1 M cacodylate buffer [pH 7.2]; 0.5% glutaraldehyde for LM, and 2.5% glutaraldehyde for TEM) for 12 to 18 hours at room temperature with rotation. TEM samples were postfixed in osmium tetroxide (1% for 1 hour at 4°C). Both were treated with uranyl acetate (2% for 1 hour at room temperature) followed by alcohol dehydration. For LM, microtome-cut thick sections were embedded in LR-White resin (EMS, Hatfield, PA), sectioned to 0.7-m thickness, and stained with toluidine blue oxide. These histologic sections were examined on a light microscope (DMR; Leica Bannockburn, IL) equipped with a 12-megapixel charge-coupled device (CCD) digital camera (DC-500; Leica). For TEM, microtome-cut thick sections were embedded in Epon epoxy resin (EMS), thin-sectioned to 70 nm with a diamond knife (Diatome; EMS), and grid stained with uranyl acetate and lead citrate. Thin sections were examined with a transmission electron microscope (FEI-Philips Tecnai 12; FEI Co., Hillsboro, OR) at 80 kV using a 1 k ϫ 1 k CCD digital camera (model 694l; Gatan, Pleasanton, CA). Protocols were reviewed by the Institutional Review Board and the research was conducted according to the tenets of the Declaration of Helsinki for the protection of human subjects. Theoretical Analysis The scattering cross section of a single MLB of radius r is equal to qr 2 , where q is the scattering efficiency of the MLB. Because the volume fraction of the MLBs in the lens is quite small (Ͻ0.005%) and they are randomly distributed, they can be considered as independent scatterers. If no MLB shadowed another, the relative scattering cross section of N MLBs would equal Nq r 2 /A where A is the cross-sectional area of the central square millimeter of the lens nucleus. In this region N is equal to tA, where is the density of MLBs (per cubic millimeter) and t is the thickness of the nucleus (along the path of incident light). Therefore, the relative scattering cross section of all the MLBs in the center of the lens nucleus would be 11 S/A ϭ tqr 2 However, the values must be corrected for the fact that each incident photon has a probability of being scattered by an MLB before it reaches another MLB deeper in the lens nucleus. This process is analogous to the exponential decrease in probability that a raindrop will hit a dry spot after previous raindrops have begun covering a dry surface. This issue has been addressed in a derivation of Beer's Law, The scattering efficiency, q, was calculated employing Mie theory implemented by using Mätzler's algorithms 16 in commercial software (MatLab; MathWorks, Natick, MA). Each point on the surface plots represents a single calculation for ranges of parameters relevant to the observed MLBs. The key input parameters were the ratios of particle diameters to wavelength and the ratios of internal refractive indices of the MLBs to cytoplasmic refractive index, measured directly to be 1.40. 14 This average scattering angle, , is given by where I() is the intensity of light scattered at the angle . For small particles, which scatter as the average scattering angle is 90°. For large particles, the average angle can be less than 10°. The relative scattering cross section, S/A, was calculated for three wavelengths of unpolarized light, given the observed MLB radii and their densities in transparent and cataractous nuclei. 11 RESULTS Morphologic Analysis Further characterization of MLBs along the optic axis are presented in Figures 1 to 3, which display new images of MLBs, emphasizing their unique features and the distribution of sizes not previously published. Light microscopy of toluidine blue oxide histologic sections reveals MLBs as circular profiles with white borders 10,11 These particles present circular profiles in sections, and, since two thirds of the particles examined here present circular profiles in sections, it is reasonable to assume that they are also spherical. The remaining one third are slightly oval or have several components, at least one of which appears to be spherical Particle dimensions for all 117 MLBs observed in the cataractous nuclei (including the embryonic, fetal and juvenile nuclei) have been presented as a frequency plot 11,20 The corrected diameter is 2.7 Ϯ 0.7 m. The distribution of particle diameters is almost normal, with the exception of a tail at larger diameters indicated by the observation of several particles more than 3.5 m in diameter. A larger number of measurements would be necessary to determine whether this minor deviation from normal distribution is significant. The shape of the frequency distribution and the Even when more than one MLB was visible in the same field of view, they were separated by distances much larger than the wavelength of incident light. The MLB at the upper right had a core visible at a different plane of focus. Edge processes (arrowheads) were numerous profiles also found with a similar density in transparent lenses. close correspondence of the mean and median (2.7 m vs. 2.6 m, respectively) suggest that the value of 2.7 m is a reasonable representation of a typical MLB diameter in the human nuclear cataracts examined. The range of particle sizes used in the Mie scattering calculations was adjusted to include the full range of particle sizes observed. From TEM images at high magnification, there appeared to be 3 to 10 thin bilayers forming the coat of the MLBs Differences between the refractive indices of the particles and the surrounding cytoplasm are essential to produce observable light scattering. TEM images clearly show that many MLBs have interiors that are very different from their surroundings 21 Although this appears to be a high refractive index for an ocular lens, it is noted that this refractive index is still somewhat less than the value of 1.55 reported for the nuclear core of fish lenses. Mie Theory Calculations of Particle Light Scattering The scattering efficiency, q, per MLB in unpolarized light of 550 nm wavelength was calculated for a range of particle sizes and refractive indices of the MLB interior, assuming that the lipid-rich coat has n ϭ 1.50 and the cytoplasm has n ϭ 1.40 This surface plot reveals the remarkable result that there is the potential for particles to have very large scattering efficiencies. Because they have such strong interactions with light at visible wavelengths, their scattering cross section is nearly three times larger than the actual particle cross section. This finding is important from another viewpoint. The surface plot suggests that relatively few super scattering particles can generate more scattered light than a much larger number of less efficient scattering particles. It should also be noted that valleys (blue regions) give valuable information. Thus, the valley at n ϭ 1.40 indicates that, regardless of particle size in the range displayed, if the interior refractive index is less than 1.42 (ratio 1.42/1.40 ϭ 1.0143), then these particles will generate very little scattered light. In addition, over the entire range of refractive indices displayed, particles with radii ranging from 0.05 to 0.3 m are extremely inefficient scatterers. Finally, particles with internal refractive indices lower than the surrounding cytoplasm scatter light with roughly the same efficiency. For the typical MLB, based on the actual particles observed and an assumed internal refractive index of 1.49, the calculated scattering efficiency was 2.6, which would place the particle near the top of the steep slope of the surface plot It has been shown in a polar plot that incident light is scattered by typical MLBs (1.36 m radius, internal n ϭ 1.49) FIGURE 3. Frequency plot of MLBs in cataractous nuclei. A total of 117 MLBs were observed in the juvenile, fetal, and embryonic nuclei of human age-related cataracts. 11 The diameter of MLBs presenting a circular profile (two thirds of all particles) was measured on light micrographs to the outer edge of the clear rim around the particle. For complex MLBs, the length and width were measured and averaged to give a diameter of an equivalent circular profile. The distribution was nearly normal, with a tail containing several particles larger than 3.5 m diameter. Mean Ϯ SD of the raw data is 2.13 Ϯ 0.64 m. with high intensity in the forward direction within a narrow cone of 20°scattering angle (i.e., Ϯ10°around the optic axis). 11 This angular dependence of scattered light is more completely displayed in the surface plot calculated using unpolarized 550-nm incident light 9,13,14 Of particular interest is the relative amount of light scatter predicted by Mie scattering theory for the measured density of MLBs reported. 11 The relative scattering cross section, S/A, is calculated for the observed MLBs (summarized in DISCUSSION Mie scattering theory is appropriate for the coated particles observed in nuclei of aged transparent lenses and age-related cataracts. The theory gives exact solutions for spherical particles of any size and refractive index, and gives fairly accurate solutions for globular particles in general. 9,12-14 The conditions for Mie scattering calculations, mainly the random distribution of globular particles separated by distances greater than the wavelength of incident light, are well met by the observed MLBs. 11 In contrast, the utility of Rayleigh scattering theory, even incorporating modifications or approximations such as Debye-Gans, 9,13,14 is limited for large particles. Wavelength dependence of Rayleigh scattering theory for independent scatterers and the predicted scattering roughly equal in all directions is suitable for small particles about one tenth of the wavelength of light, 9,13,14 but cannot predict correctly the low-angle scattering or the scattering efficiency of the MLBs. The algorithms for Mie scattering theory permit the calculation of three-dimensional surface plots describing potential scattering from coated spherical particles over a wide range of sizes, refractive index properties and wavelengths of incident light. 308 Costello et al. IOVS, January 2007, Vol. 48, No. 1 Downloaded from iovs.arvojournals.org on 06/28/2019 FIGURE 7. Surface plots of percentage light scatter (rainbow scales, 0%-70%) as a function of particle radius and refractive index of the particle interior. Predicted scattering from MLBs is based on observed density in transparent nuclei (A-C) and cataractous nuclei (D-F) Fiber cell membranes have also been implicated in cataract formation through direct scattering of membrane components, alterations of membranes and deposition of proteins on membranes. Recently, Michael et al., 11,29 An additional factor is that the scattering from the membranes is dependent mainly on the membrane thickness rather than the membrane topology. Minimal light is scattered by sheets that are thin compared with the wavelength of incident light, even if their topology is complex. The surface plots highlight the features of particles responsible for forward scattering and give direct visualization of the scattering performance of experimentally observed MLBs. Although the observed MLBs displayed a range of particle sizes, for simplicity, the average diameter measured in sections was used for the Mie calculations. The critical property of the ratio of internal to cytoplasmic refractive indices (1.49/1.40) was chosen to illustrate the effect of having particles that are significantly different as indicated from electron micrographs

Novel mitochondrial derived Nuclear Excisosome degrades nuclei during differentiation of prosimian Galago (bush baby) monkey lenses.

The unique cellular organization and transparent function of the ocular lens depend on the continuous differentiation of immature epithelial cells on the lens anterior surface into mature elongated fiber cells within the lens core. A ubiquitous event during lens differentiation is the complete elimination of organelles required for mature lens fiber cell structure and transparency. Distinct pathways have been identified to mediate the elimination of non-nuclear organelles and nuclei. Recently, we reported the discovery of a unique structure in developing fiber cells of the chick embryo lens, called the Nuclear Excisosome, that is intractably associated with degrading nuclei during lens fiber cell differentiation. In the chick lens, the Nuclear Excisosome is derived from projections of adjacent cells contacting the nuclear envelope during nuclear elimination. Here, we demonstrate that, in contrast to the avian model, Nuclear Excisosomes in a primate model, Galago (bush baby) monkeys, are derived through the recruitment of mitochondria to form unique linear assemblies that define a novel primate Nuclear Excisosome. Four lenses from three monkeys aged 2-5 years were fixed in formalin, followed by paraformaldehyde, then processed for Airyscan confocal microscopy or transmission electron microscopy. For confocal imaging, fluorescent dyes labelled membranes, carbohydrate in the extracellular space, filamentous actin and nuclei. Fiber cells from Galago lenses typically displayed prominent linear structures within the cytoplasm with a distinctive cross-section of four membranes and lengths up to 30 μm. The outer membranes of these linear structures were observed to attach to the outer nuclear envelope membrane to initiate degradation near the organelle-free zone. The origin of these unique structures was mitochondria in the equatorial epithelium (not from plasma membranes of adjacent cells as in the chick embryo model). Early changes in mitochondria appeared to be the collapse of the cristae and modification of one side of the mitochondrial outer membrane to promote accumulation of protein in a dense cluster. As a mitochondrion surrounded the dense protein cluster, an outer mitochondrial membrane enclosed the protein to form a core and another outer mitochondrial membrane formed the outermost layer. The paired membranes of irregular texture between the inner core membrane and the outer limiting membrane appeared to be derived from modified mitochondrial cristae. Several mitochondria were involved in the formation and maturation of these unique complexes that apparently migrated around the fulcrum into the cytoplasm of nascent fiber cells where they were stabilized until the nuclear degradation was initiated. Thus, unlike in the chick embryo, the Galago lenses degraded nuclear envelopes with a Nuclear Excisosome derived from multiple mitochondria in the epithelium that formed novel linear assemblies in developing fiber cells. These findings suggest that recruitment of distinct structures is required for Nuclear Excisosome formation in different species