Effects of explant size on epithelial outgrowth, thickness, stratification, ultrastructure and phenotype of cultured limbal epithelial cells.

PURPOSE: Transplantation of limbal stem cells is a promising therapy for limbal stem cell deficiency. Limbal cells can be harvested from either a healthy part of the patient's eye or the eye of a donor. Small explants are less likely to inflict injury to the donor site. We investigated the effects of limbal explant size on multiple characteristics known to be important for transplant function. METHODS: Human limbal epithelial cells were expanded from large versus small explants (3 versus 1 mm of the corneal circumference) for 3 weeks and characterized by light microscopy, immunohistochemistry, and transmission electron microscopy. Epithelial thickness, stratification, outgrowth, ultrastructure and phenotype were assessed. RESULTS: Epithelial thickness and stratification were similar between the groups. Outgrowth size correlated positively with explant size (r = 0.37; P = 0.01), whereas fold growth correlated negatively with explant size (r = -0.55; P < 0.0001). Percentage of cells expressing the limbal epithelial cell marker K19 was higher in cells derived from large explants (99.1±1.2%) compared to cells derived from small explants (93.2±13.6%, P = 0.024). The percentage of cells expressing ABCG2, integrin ß1, p63, and p63α that are markers suggestive of an immature phenotype; Keratin 3, Connexin 43, and E-Cadherin that are markers of differentiation; and Ki67 and PCNA that indicate cell proliferation were equal in both groups. Desmosome and hemidesmosome densities were equal between the groups. CONCLUSION: For donor- and culture conditions used in the present study, large explants are preferable to small in terms of outgrowth area. As regards limbal epithelial cell thickness, stratification, mechanical strength, and the attainment of a predominantly immature phenotype, both large and small explants are sufficient.

Precision Measurement of the First Ionization Potential of Nobelium.

One of the most important atomic properties governing an element's chemical behavior is the energy required to remove its least-bound electron, referred to as the first ionization potential. For the heaviest elements, this fundamental quantity is strongly influenced by relativistic effects which lead to unique chemical properties. Laser spectroscopy on an atom-at-a-time scale was developed and applied to probe the optical spectrum of neutral nobelium near the ionization threshold. The first ionization potential of nobelium is determined here with a very high precision from the convergence of measured Rydberg series to be 6.626 21±0.000 05 eV. This work provides a stringent benchmark for state-of-the-art many-body atomic modeling that considers relativistic and quantum electrodynamic effects and paves the way for high-precision measurements of atomic properties of elements only available from heavy-ion accelerator facilities.

Effect of Transportation on Cultured Limbal Epithelial Sheets for Worldwide Treatment of Limbal Stem Cell Deficiency.

Limbal stem cell deficiency can be treated with transplantation of cultured human limbal epithelial cells (LEC). It can be advantageous to produce LEC in centralized labs and thereafter ship them to eye clinics. The present study used transport simulations of LEC to determine if vigorous shaking during transport altered the viability, morphology and phenotype during a 4 day-long storage of LEC with a previously described serum-free storage method. Inserts with LEC cultured on amniotic membranes were sutured to caps inside air-tight containers with generous amounts of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered minimal essential medium (MEM). The containers were distributed among the following testing conditions: 6 hours with full containers, 36 hours with full containers, 36 hours with container three quarters full of medium, and 36 hours with container full of medium containing a shear-protecting agent (Pluronic-F68). Compared to stored, but non-transported controls, no statistically significant changes in viability and immunohistochemical staining were observed. The epithelial sheets remained intact. However, an air-liquid interface in the containers reduced the number of desmosomes and hemi-desmosomes compared to the controls. In conclusion, cultured LEC sheets appear to endure vigorous shaking for at least 36 hours if the container is full.

Probing Sizes and Shapes of Nobelium Isotopes by Laser Spectroscopy.

Until recently, ground-state nuclear moments of the heaviest nuclei could only be inferred from nuclear spectroscopy, where model assumptions are required. Laser spectroscopy in combination with modern atomic structure calculations is now able to probe these moments directly, in a comprehensive and nuclear-model-independent way. Here we report on unique access to the differential mean-square charge radii of ^{252,253,254}No, and therefore to changes in nuclear size and shape. State-of-the-art nuclear density functional calculations describe well the changes in nuclear charge radii in the region of the heavy actinides, indicating an appreciable central depression in the deformed proton density distribution in ^{252,254}No isotopes. Finally, the hyperfine splitting of ^{253}No was evaluated, enabling a complementary measure of its (quadrupole) deformation, as well as an insight into the neutron single-particle wave function via the nuclear spin and magnetic moment.

Towards high-resolution laser ionization spectroscopy of the heaviest elements in supersonic gas jet expansion.

Resonant laser ionization and spectroscopy are widely used techniques at radioactive ion beam facilities to produce pure beams of exotic nuclei and measure the shape, size, spin and electromagnetic multipole moments of these nuclei. However, in such measurements it is difficult to combine a high efficiency with a high spectral resolution. Here we demonstrate the on-line application of atomic laser ionization spectroscopy in a supersonic gas jet, a technique suited for high-precision studies of the ground- and isomeric-state properties of nuclei located at the extremes of stability. The technique is characterized in a measurement on actinium isotopes around the N=126 neutron shell closure. A significant improvement in the spectral resolution by more than one order of magnitude is achieved in these experiments without loss in efficiency.

Mental Imagery-Based Training to Modify Mood and Cognitive Bias in Adolescents: Effects of Valence and Perspective.

Mental imagery has a powerful impact on emotion and cognitive processing in adults, and is implicated in emotional disorders. Research suggests the perspective adopted in mental imagery modulates its emotional impact. However, little is known about the impact of mental imagery in adolescence, despite adolescence being the key time for the onset of emotional dysfunction. We administered computerised positive versus mixed valence picture-word mental imagery training to male adolescent participants (N = 60, aged 11-16 years) across separate field and observer perspective sessions. Positive mood increased more following positive than mixed imagery; pleasantness ratings of ambiguous pictures increased following positive versus mixed imagery generated from field but not observer perspective; negative interpretation bias on a novel scrambled sentences task was smaller following positive than mixed imagery particularly when imagery was generated from field perspective. These findings suggest positive mental imagery generation alters mood and cognition in male adolescents, with the latter moderated by imagery perspective. Identifying key components of such training, such as imagery perspective, extends understanding of the relationship between mental imagery, mood, and cognition in adolescence.

Breakdown of the isobaric multiplet mass equation for the A = 20 and 21 multiplets.

Using the Penning trap mass spectrometer TITAN, we performed the first direct mass measurements of (20,21)Mg, isotopes that are the most proton-rich members of the A = 20 and A = 21 isospin multiplets. These measurements were possible through the use of a unique ion-guide laser ion source, a development that suppressed isobaric contamination by 6 orders of magnitude. Compared to the latest atomic mass evaluation, we find that the mass of (21)Mg is in good agreement but that the mass of (20)Mg deviates by 3 σ. These measurements reduce the uncertainties in the masses of (20,21)Mg by 15 and 22 times, respectively, resulting in a significant departure from the expected behavior of the isobaric multiplet mass equation in both the A = 20 and A = 21 multiplets. This presents a challenge to shell model calculations using either the isospin nonconserving universal sd USDA and USDB Hamiltonians or isospin nonconserving interactions based on chiral two- and three-nucleon forces.

Ion source developments for the production of radioactive isotope beams at TRIUMF.

At the ISAC facility at TRIUMF radioactive ions are produced by bombarding solid targets with up to 100 µA of 500 MeV protons. The reaction products have to diffuse out of the hot target into an ion source. Normally, singly charged ions are extracted. They can be transported either directly to experiments or via an ECR charge state breeder to a post accelerator. Several different types of ion sources have to be used in order to deliver a large variety of rare isotope beams. At ISAC those are surface ion sources, forced electron beam arc discharge (FEBIAD) ion sources and resonant laser ionization sources. Recent development activities concentrated on increasing the selectivity for the ionization to suppress isobaric contamination in the beam. Therefore, a surface ion rejecting resonant laser ionization source (SIRLIS) has been developed to suppress ions from surface ionization. For the FEBIAD ion source a cold transfer line has been introduced to prevent less volatile components from reaching the ion source.

Measurement of the first ionization potential of astatine by laser ionization spectroscopy.

The radioactive element astatine exists only in trace amounts in nature. Its properties can therefore only be explored by study of the minute quantities of artificially produced isotopes or by performing theoretical calculations. One of the most important properties influencing the chemical behaviour is the energy required to remove one electron from the valence shell, referred to as the ionization potential. Here we use laser spectroscopy to probe the optical spectrum of astatine near the ionization threshold. The observed series of Rydberg states enabled the first determination of the ionization potential of the astatine atom, 9.31751(8) eV. New ab initio calculations are performed to support the experimental result. The measured value serves as a benchmark for quantum chemistry calculations of the properties of astatine as well as for the theoretical prediction of the ionization potential of superheavy element 117, the heaviest homologue of astatine.

Biopsy harvesting site and distance from the explant affect conjunctival epithelial phenotype ex vivo.

The purpose of the study was to investigate if the number of goblet cells expanded ex vivo from a conjunctival explant is affected by the biopsy harvesting site on the conjunctiva and the distance from the explant. Conjunctival explants from six regions: superior and inferior bulbus, fornix, and tarsus of male Sprague-Dawley rats were grown in RPMI 1640 with 10% fetal bovine serum on coverslips for eight days. Histochemical and immunofluorescent staining of goblet (CK-7/UEA-1/MUC5AC), stratified squamous, non-goblet (CK-4), proliferating (PCNA) and progenitor (ABCG2) cells were analyzed by epifluorescence and laser confocal microscopy. Outgrowth was measured with NIH ImageJ. For statistical analysis the Mann-Whitney test and Spearman's rank-order correlation test were used. Cultures from superior and inferior fornix contained the most goblet cells as indicated by the presence of CK-7+, UEA-1+ and MUC5AC+ cells. Superior and inferior forniceal cultures displayed 60.8% ± 9.2% and 64.7% ± 6.7% CK-7+ cells, respectively, compared to the superior tarsal (26.6% ± 8.4%; P < 0.05), superior bulbar (31.0% ± 4.0%; P < 0.05), inferior bulbar (38.5% ± 9.3%; P < 0.05) and inferior tarsal cultures (27.7% ± 8.3%; P < 0.05). While 28.4% ± 6.3% of CK-7+ goblet cells co-labeled with PCNA, only 7.4% ± 1.6% of UEA-1+ goblet cells did (P < 0.01). CK-7+ goblet cells were located at a lower concentration close to the explant (39.8% ± 3.1%) compared to near the leading edge (58.2% ± 4.5%; P < 0.05). Both markers for goblet cell secretory product (UEA-1 and MUC5AC), however, displayed the opposite pattern with a higher percentage of positive cells close to the explant than near the leading edge (P < 0.05). The percentage of CK-4+ cells was higher near the explant compared to near the leading edge (P < 0.01). The percentage of CK-7+ goblet cells in the cultures did not correlate with the outgrowth size (r(s) = -0.086; P = 0.435). The percentage of UEA-1+ goblet cells correlated negatively with outgrowth size (r(s) = -0.347; P < 0.01), whereas the percentage of CK-4+ cells correlated positively with the outgrowth size (r(s) = 0.473; P < 0.05). We conclude that forniceal explants yield the highest number of goblet cells ex vivo and thereby seem to be optimal for goblet cell transplantation. We also suggest that CK-7+/UEA-1- cells represent highly proliferative immature goblet cells. These cells could be important during conjunctival migration as they are mostly located close to the leading edge and their density does not decrease with increasing outgrowth size.

Detection of plutonium isotopes at lowest quantities using in-source resonance ionization mass spectrometry.

The in-source resonance ionization mass spectrometry technique was applied for quantification of ultratrace amounts of plutonium isotopes as a proof of principle study. In addition to an overall detection limit of 10(4) to 10(5) atoms, this method enables the unambiguous identification and individual quantification of the plutonium isotopes (238)Pu and (241)Pu which are of relevance for dating of radiogenic samples. Due to the element-selective ionization process, these isotopes can be measured even under a high surplus of isobaric contaminations from (238)U or (241)Am, which considerably simplifies chemical preparation. The technique was developed, tested, and characterized on a variety of synthetic and calibration samples and is presently applied to analyze environmental samples.

Progress of ultra trace determination of technetium using laser resonance ionization mass spectrometry.

Laser resonance ionization mass spectrometry (RIMS) represents one of the most sensitive and selective techniques for ultra trace determination of long-lived radioisotopes. The isotope (99g)Tc constitutes a specific candidate of high relevance concerning its environmental behavior as well as fundamental research applications. Based on the recent precision determination of the ionization potential of technetium by laser resonance ionization, refined resonant optical excitation pathways have been derived for analytical determination of ultra trace amounts of (99g)Tc by laser mass spectrometric approaches. The state of the art and the specifications of RIMS-based ultra trace determination for (99g)Tc, leading to a level of detection of ε ≈ 3 × 10(-4) atoms (3 µBq), are reported.

Effects of serum-free storage on morphology, phenotype, and viability of ex vivo cultured human conjunctival epithelium.

The use of amniotic membrane (AM) represents one of the major developments in ocular surface reconstruction. However, in a study on patients with primary pterygium, transplantation of AM with ex vivo expanded human conjunctival epithelial cells (HCjE) promoted earlier epithelialization than AM alone. We previously showed that cultured human limbal epithelial cells maintain their morphology, phenotype, and viability for one week when stored at 23°C. The current study investigates the feasibility of storing HCjE in HEPES-MEM and Optisol-GS at 23°C for 4 and 7 days, respectively. The five experimental groups were analyzed by light microscopy, immunohistochemistry, transmission electron microscopy, and a viability assay. The ultrastructural integrity of cultured HCjE was well preserved following 4 days of storage, however, 7 days of storage resulted in some loss of cell-cell contacts and epithelial detachment from the amniotic membrane. The number of microvilli in cultured HCjE not subjected to storage was 2.03±0.38 microvilli/µm. In comparison, after 4 and 7 days of HEPES-MEM storage this number was 1.69±0.54 microvilli/µm; P=0.98 and 0.89±1.0 microvilli/µm; P=0.28, respectively. After Optisol-GS storage for 4 and 7 days, the mean number of microvilli was 1.07±1.0 microvilli/µm; P=0.47 and 0.07±0.07 microvilli/µm; P=0.03, respectively. The number of cell layers in cultured HCjE not subjected to storage was 4.4±0.3 cell layers, as opposed to 4.0±0.9 cell layers; P=0.89 after 4 days of HEPES-MEM storage and 2.8±0.6 cell layers; P=0.01 after 7 days of storage in HEPES-MEM. The number of cell layers after 4 and 7 days of storage in Optisol-GS was 3.7±0.2 cell layers; P=0.46 and 3.4±0.4 cell layers; P=0.18, respectively. The expression of markers for undifferentiated cells (ΔNp63α, ABCG2 and p63), proliferating cells (Ki67 and PCNA), goblet cells (Ck7 and MUC5AC), stratified squamous epithelial cells (Ck4), and apoptotic cells (caspase-3) in cultured HCjE appeared to be unchanged after 4 and 7 days of HEPES-MEM and Optisol-GS storage. The percentage of viable cells in cultured HCjE not subjected to storage (91.4%±3.2%) was sustained after 4 and 7 days of storage in HEPES-MEM (94.1%±4.5%; P=0.99 and 85.1%±13.7%; P=0.87, respectively) as well as after 4 and 7 days of storage in Optisol-GS (87.7%±15.2%; P=0.97 and 79.8%±15.7%; P=0.48, respectively). We conclude that cultured HCjE may be stored for at least 4 days in serum-free conditions at 23°C while maintaining the phenotype and viability. HEPES-MEM appears to be comparable to Optisol-GS for serum-free storage with preservation of the ultrastructure for at least 4 days.

Ion production from solid state laser ion sources.

Laser ion sources based on resonant excitation and ionization of atoms are well-established tools for selective and efficient production of radioactive ion beams. Recent developments are focused on the use of the state-of-the-art all solid-state laser systems. To date, 35 elements of the periodic table are available from laser ion sources based on tunable Ti:sapphire lasers. Recent progress in this field regarding the establishment of suitable optical excitation schemes for Ti:sapphire lasers are reported.

Production of negative osmium ions by laser desorption and ionization.

The interest to produce negative osmium ions is manifold in the realm of high-accuracy ion trap experiments: high-resolution nearly Doppler-free laser spectroscopy, antihydrogen formation in its ground state, and contributions to neutrino mass spectrometry. Production of these ions is generally accomplished by sputtering an Os sample with Cs(+) ions at tens of keV. Though this is a well-established method commonly used at accelerators, these kind of sources are quite demanding and tricky to operate. Therefore, the development of a more straightforward and cost effective production scheme will be of benefit for ion trap and other experiments. Such a scheme makes use of desorption and ionization with pulsed lasers and identification of the ions by time-of-flight mass spectrometry. First investigations of negative osmium ion production using a pulsed laser for desorption and ionization and a commercial matrix-assisted laser desorption/ionization time-of-flight system for identification has demonstrated the suitability of this technique. More than 10(3) negative osmium ions per shot were registered after bombarding pure osmium powder with a 5 ns pulse width Nd:yttrium aluminum garnet laser. The limitation in the ion number was imposed by the detection limit of the microchannel plate detector.

Sterility control and long-term eye-bank storage of cultured human limbal epithelial cells for transplantation.

BACKGROUND/AIMS: To assess sterility of cultured human limbal epithelial cells (HLEC) and to investigate the viability, morphology and phenotype of cultured HLEC following 2 and 3 weeks of organ culture storage. METHODS: HLEC cultured on amniotic membranes were stored in organ culture medium in a closed container at 23 degrees C. Sterility of storage media was tested using a Bactec 9240 blood culture instrument (Becton Dickinson, Maryland) for incubation and periodic reading. Viability was analysed by calcein-acetoxymethyl ester/ethidium homodimer-1 assay, morphology by light microscopy and cellular phenotype by immunohistochemistry. RESULTS: No microbial contamination was observed after 1 week's storage. Viability of cultured HLEC was 87.9 (SD 6.4)% and 52.7 (13.1)% after 2 and 3 weeks of storage, respectively, compared with 98.8 (2.6)% before storage (p<0.001). The multilayered structure was preserved in 70% of cultures following 2 weeks of storage but lost after 3 weeks. A less differentiated phenotype was maintained. CONCLUSION: This study is the first to verify the sterility of HLEC cultures prior to transplantation. Although a slight decrease in viability was observed following 2 weeks of storage, the HLEC sheets remain acceptable, whereas 3 week's storage was unsatisfactory.

Homocysteine measurements in geriatric patients.

OBJECTIVE: Homocysteine measurements may be relevant in geriatric medicine as homocysteine has been identified as an independent risk factor for prevalent disorders such as occlusive arterial vascular disease, cognitive impairment and dementia. The aim of the present study was to study diagnostic correlates of plasma total homocysteine (tHcy) in geriatric in-patients. MATERIAL AND METHODS: Blood samples for the analysis of tHcy and related factors like serum vitamin B12, serum folate, red blood cell folate and clinical data were collected from geriatric patients (n=114) in stable clinical condition. RESULTS: Almost 40% of the patients had tHcy values above 20 micromol/L. tHcy correlated significantly with serum folate, serum vitamin B12, serum creatinine and congestive heart failure, but not with red blood cell folate, cerebrovascular disease, coronary heart disease or cognitive impairment. CONCLUSIONS: Hyperhomocysteinaemia seems to be frequent in geriatric patients and might primarily be an indicator of low folate and high creatinine values.