Corneal Endothelium Viability Assay Using Trypan Blue Dye After Preloaded Descemet Membrane Endothelial Keratoplasty Graft Preparation.

PURPOSE: The purpose of this study was to establish a validated method, consistent with Eye Bank Association of America medical standards, for evaluating endothelial cell loss (ECL) from an entire Descemet membrane endothelial keratoplasty (DMEK) graft using trypan blue dye as an alternative to specular microscopy. METHOD: Twenty-nine corneas were prepared for preloaded DMEK by a single technician, and the endothelium was stained with trypan blue dye for 30 seconds. The technician estimated total cell loss as a percentage of the graft and captured an image. Images were evaluated by a blinded technician using ImageJ software to determine ECL and compared with endothelial cell density from specular microscopy. Tissue processing intervals were analyzed for 4 months before and after implementation of this method. RESULTS: For the 29 grafts, there was no statistically significant difference ( t test, P = 0.285) between ECL estimated by a processor (mean = 5.8%) and ECL calculated using an ImageJ software (mean = 5.1%). The processor tended to estimate greater ECL than the actual ECL determined by ImageJ (paired t test, P = 0.022). Comparatively, postprocessing endothelial cell density measured by specular microscopy were higher compared with the preprocessing endothelial cell density (mean = 4.5% P = 0.0006). After implementation of this evaluation method, DMEK graft processing time intervals were reduced by 47.9% compared with specular microscopy evaluation ( P < 0.001). CONCLUSIONS: Our results show that visual ECL estimation using trypan blue staining by a DMEK graft processor is a reliable and efficient method for endothelial assessment. Unlike specular microscopy, this method achieves comprehensive visualization of the entire endothelium, reduces total time out of cold storage, and decreases total time required to prepare and evaluate DMEK grafts.

COVID-19 and Eye Banking: Ongoing Impacts of the Pandemic.

PURPOSE: The purpose of this study was to assess the impact of ongoing waves of the COVID-19 pandemic and resulting guidelines on the corneal donor pool with resumption of clinical operations. METHODS: A retrospective analysis of donors deemed eligible for corneal transplantation at an eye bank from July 1, 2020, through December 31, 2021. Donors ineligible due to meeting Eye Bank Association of America (EBAA) COVID-19 guidelines or a positive postmortem COVID-19 testing were examined. The correlation between COVID-19 rule outs and state COVID positivity was calculated. The number of scheduled surgeries, suitable corneas, imports, and international exports was compared with a pre-COVID period. Postmortem testing was reduced for the final 5 months of the study, and numbers were compared before and after the policy change. RESULTS: 2.85% of referrals to the eye bank were ruled out because of EBAA guidelines. 3.2% of postmortem tests were positive or indeterminate resulting in an ineligible tissue donor (0.42% of referrals). Over the 18-month period, there was a 4.30% shortage of suitable corneas compared with transplantation procedures. There was a significant correlation between postmortem testing and state COVID-19 positivity (r = 0.37, P <0.01), but not with EBAA guidelines (r = 0.19, P = 0.07). When postmortem testing was reduced, significantly more corneas were exported internationally. CONCLUSIONS: Although corneal transplant procedures were back to normal levels, there was a shortage of suitable corneal tissue. The discontinuation of postmortem testing was associated with a significant increase in international exports of corneal donor tissue.

Effect of Increasing Povidone-Iodine Exposure on Corneal Epithelium and Impact on Donor Rim Cultures.

PURPOSE: The purpose of this study was to assess the effect of a second povidone-iodine (PVP-I) application at the time of donor tissue recovery on overall tissue quality and to analyze the rate of positive fungal and bacterial rim cultures before and after implementing increased PVP-I exposure. METHODS: The left cornea was recovered after a single application of PVP-I, while the right cornea was recovered after double PVP-I application in research-consented donors. The epithelial cell death rate was estimated using viability assay in corneal whole mounts under 10× objective (n = 5). Clinical characteristics of epithelium, stroma, and endothelium; positive rim culture rate; and incidences of infectious postoperative adverse reactions were compared for a period of 14 months before and after implementation of increased PVP-I protocol. RESULTS: The average epithelial cell death rate was unaltered between single and double PVP-I exposure groups. We observed a modest 10% increase in the number of tissues with mild edema after implementation of increased PVP-I exposure. Nonetheless, the percentage of tissues with moderate or severe edema was unaltered. The average positive rim culture rate decreased from 1.17% to 0.88% (P = 0.075) after implementation of the double PVP-I soak procedure. There has been only one report of infectious postoperative adverse reactions since this procedure change. By contrast, there were 5 reports for a period of 14 months before implementation of this protocol. CONCLUSIONS: These results indicate that new donor preparation methods with an additional 5 minutes of PVP-I exposure do not affect tissue quality, reduce positive rim cultures, and lead to lower incidence of postoperative infection.

Comparison of Graft Outcomes Reusing Original Intermediate-Term Cold Storage Solution for Entire Corneal Donor Storage Period With Exchanged Fresh Storage Solution After Donor Preparation in the Cornea Preservation Time Study.

PURPOSE: The purpose of this study was to evaluate outcomes of Descemet stripping automated endothelial keratoplasty comparing exchange with fresh intermediate-term cold storage solution after lenticule preparation versus reuse of the original solution in the Cornea Preservation Time Study. METHODS: In the Cornea Preservation Time Study, 508 donor corneas had lenticules prepared by the eye bank with fresh solution exchange (Fresh group) and 283 with reuse of the original solution (Original group). Storage time for all donors was ≤11 days. Graft success rates, central endothelial cell loss at 3 years, and frequency of positive donor rim cultures were compared between the 2 groups. RESULTS: The 3-year graft success rate (95% confidence interval) was 93.4% (90.7%-95.3%) in the Fresh group and 95.2% (91.8%-97.2%) in the Original group (adjusted hazard ratio for graft failure = 0.64, 95% confidence interval, 0.33-1.24, P = 0.19). The mean percentage endothelial cell loss was significantly greater in the Fresh group versus Original group (45% ± 22% vs. 38% ± 20%, respectively, P = 0.004). Cultures were positive in 4 (1.5%) of 267 donor rims (3 fungal and 1 bacterial) in the Fresh group and in 4 (2.5%) of 158 in the Original group ( P = 0.57). There were 2 postoperative infections in the Original group and none in the Fresh group. CONCLUSIONS: The use of the original intermediate-term cold storage solution did not reduce the 3-year graft success rate compared with exchanging with fresh solution after lenticule preparation for Descemet stripping automated endothelial keratoplasty, while the frequency of positive donor rim cultures did not significantly differ between groups.

Trends in Early Graft Failure Leading to Regrafting After Endothelial Keratoplasty in the United States.

PURPOSE: The purpose of this study was to report trends in the prevalence of early graft failure after endothelial keratoplasty in the United States. METHODS: Descemet membrane endothelial keratoplasty (DMEK) and Descemet stripping automated endothelial keratoplasty (DSAEK) graft volumes were collected from records maintained by 6 major eye banks in the United States from January 1, 2013, to December 31, 2018. The prevalence and presumed cause of early graft failures (defined as a graft with persistent edema or regrafted within 8 weeks after keratoplasty) each year were sourced from surgeon-reported adverse events. Failed graft cases from the 3 eye banks were compared with nonfailures at the donor and recipient levels to perform subset analysis of factors associated with early graft failure. RESULTS: A total of 51,887 endothelial keratoplasty tissues were distributed during the study period; 72% were DSAEK grafts. The total number of early graft failures reported was 168 of 14,284 (1.18%) for DMEK and 322 of 37,603 (0.86%) for DSAEK. Early DMEK failures decreased from 2013 (7.69%) to 2018 (0.68%). In generalized linear mixed model analyses adjusting for donor tissue characteristics, recipient age, and diagnosis, an association of borderline significance was found between higher donor age and early failure [odds ratio (95% confidence interval): 1.03 (1.00-1.05); unit change of 1 yr] and DSAEK [odds ratio 1.02 (1.00-1.04); unit of change 1 yr] cases. CONCLUSIONS: The proportion of early graft failures in DMEK decreased over time and was comparable with failure rates in DSAEK at the end of the study period. The surgical learning curve might have played a role.

Comparison of Donor Corneal Endothelial Cell Density Determined by Eye Banks and by a Central Image Analysis Reading Center Using the Same Image Analysis Method.

PURPOSE: The purpose of this study was to evaluate agreement between eye banks (EBs) and an image analysis reading center on endothelial cell density (ECD) determinations using the same image analysis method. METHODS: The Cornea Image Analysis Reading Center (CIARC) determined ECD with a single experienced analyst on EB-obtained central endothelial images from donors intended for keratoplasty from 2 eye banks, Eversight and Lions VisionGift, using the Konan center analysis method. The EBs performed ECD determination on their respective sets of images using the same analysis method with experienced eye bank technicians. RESULTS: The mean age of the 200 donors was 54 years (range 30-75 years). Seventy (35%) of the 200 patients were women, and 57 (29%) were diabetic. The mean ECD was 10 cells/mm2 greater by the EBs than by CIARC (P = 0.39), with 95% limits of agreement of [-304 to 323 cells/mm2]. The mean difference was not substantially changed when the difference between EBs and CIARC ECD was adjusted for sex, donor age, donor diabetes, CV, HEX, number of cells analyzed, and EBs as a random effect (estimated mean difference of 20 cells/mm2 after adjustment in a linear mixed model; P = 0.73). The EB-determined preoperative ECD was within 10% of the CIARC-determined ECD for 178 (89%) image sets, with 15 (8%) higher by >10% and 7 (3%) lower by >10%. CONCLUSIONS: Well-trained eye bank technicians achieve comparable results for ECD determination with an experienced image analyst from an image analysis reading center when the same image analysis method is used.

Impact of the COVID-19 Pandemic on Keratoplasty and Corneal Eye Banking.

PURPOSE: The purpose of this study was to assess the impact of COVID-19 guidelines for corneal donor tissue screening and the utility of routine postmortem COVID-19 testing of donors intended for surgical use at a single eye bank. METHODS: A retrospective analysis of referrals to and eligible donors from an eye bank between March 1, 2020, and June 30, 2020, was performed, with the same time period in 2019 as a control. Referrals who were not procured because of Eye Bank Association of America COVID-19 guidelines and eye bank-specific restrictions were noted. The results of 1 month of routine postmortem testing performed by the eye bank were examined. Analysis of variance tests were performed to assess the change between donors from 2019 to 2020. RESULTS: There was a significant reduction in both the number of total referrals to the eye bank (P = 0.044) and donors eligible for surgical transplantation (P = 0.031). Eye Bank Association of America COVID-19 guidelines reduced the number of referrals over this period by 4% to 14%. Of the 266 surgically eligible donors who received postmortem COVID-19 testing in June by the eye bank, 13 resulted positive (4.9%). CONCLUSIONS: Despite a reduction in referrals and eligible corneal transplant donors at a single eye bank, there was a surplus of surgically suitable corneal tissue during the first wave of the COVID-19 pandemic. Eye banks should consider routine postmortem COVID-19 testing to identify asymptomatic infected donors although the risk of transmission of COVID-19 from infected donors is unknown.

Patient-Reported Outcomes After Corneal Transplantation.

PURPOSE: To characterize vision-related quality of life after penetrating keratoplasty (PKP), deep anterior lamellar keratoplasty (DALK), Descemet stripping automated endothelial keratoplasty (DSAEK), and Descemet membrane endothelial keratoplasty (DMEK) using the National Eye Institute Visual Function Questionnaire (NEI-VFQ 9). METHODS: Using the Sight Outcomes Research Collaborative ophthalmology electronic health record repository, questionnaire responses were obtained from 103 PKP patients, 24 DALK patients, 42 DSAEK patients, and 50 DMEK patients undergoing postoperative examination. No exclusions were made based on preoperative diagnosis, age, complications, or comorbidities. Associations between clinical characteristics and vision-related quality of life were analyzed using nonparametric and linear regression methods. RESULTS: Patients were surveyed an average of 1.5 years postoperatively (range 24 d to 4.4 yrs). Participants who had undergone DALK, DMEK, DSAEK, and PKP had median composite VFQ scores of 77.8, 84.2, 76.1, and 70.6, respectively (P= 0.002). There were no significant differences in VFQ scores between patients treated with DMEK versus DSAEK (P = 0.440) or between patients treated with PKP versus DALK (P = 1.000). Higher postoperative acuities in the operative and fellow eyes were associated with higher VFQ scores (P < 0.001 and P < 0.001). When controlling for postoperative acuity by regression modeling, surgery type was not associated with patient-reported composite VFQ scores. CONCLUSIONS: In this study, patient-reported vision-related quality of life was similar among DMEK and DSAEK participants and also among DALK and PKP participants. When controlling for postoperative acuity, vision-related quality of life was similar among all study participants, irrespective of the keratoplasty technique.

Prevalence of SARS-CoV-2 in human post-mortem ocular tissues.

BACKGROUND: SARS-CoV-2 is found in conjunctival swabs and tears of COVID-19 patients. However, the presence of SARS-CoV-2 has not been detected in the human eye to date. We undertook this study to analyze the prevalence of SARS-CoV-2 in human post-mortem ocular tissues. METHODS: The expression of SARS-CoV-2 RNA was assessed by RT-PCR in corneal and scleral tissues from 33 surgical-intended donors who were eliminated from a surgical use per Eye Bank Association of America (EBAA) donor screening guidelines or medical director review or positive COVID-19 test. Ocular levels of SARS-CoV-2 RNA (RT-PCR), Envelope and Spike proteins (immunohistochemistry) and anti-SARS-CoV-2 IgG and IgM antibodies (ELISA) in blood were evaluated in additional 10 research-intent COVID-19 positive donors. FINDINGS: Of 132 ocular tissues from 33 surgical-intended donors, the positivity rate for SARS-CoV-2 RNA was ~13% (17/132). Of 10 COVID-19 donors, six had PCR positive post-mortem nasopharyngeal swabs whereas eight exhibited positive post-mortem anti-SARS-CoV-2 IgG levels. Among 20 eyes recovered from 10 COVID-19 donors: three conjunctival, one anterior corneal, five posterior corneal, and three vitreous swabs tested positive for SARS-CoV-2 RNA. SARS-CoV-2 spike and envelope proteins were detected in epithelial layer of the corneas that were procured without Povidone-Iodine (PVP-I) disinfection. INTERPRETATIONS: Our study showed a small but noteworthy prevalence of SARS-CoV-2 in ocular tissues from COVID-19 donors. These findings underscore the criticality of donor screening guidelines, post-mortem nasopharyngeal PCR testing and PVP-I disinfection protocol to eliminate any tissue harboring SARS-CoV-2 being used for corneal transplantation.

Prevalence of SARS-CoV-2 in human post-mortem ocular tissues.

BACKGROUND: SARS-CoV-2 is found in conjunctival swabs and tears of COVID-19 patients. However, the presence of SARS-CoV-2 has not been detected in the human eye to date. We undertook this study to analyze the prevalence of SARS-CoV-2 in human post-mortem ocular tissues. METHODS: The expression of SARS-CoV-2 RNA was assessed by RT-PCR in corneal and scleral tissues from 33 surgical-intended donors who were eliminated from a surgical use per Eye Bank Association of America (EBAA) donor screening guidelines or medical director review or positive COVID-19 test. Ocular levels of SARS-CoV-2 RNA (RT-PCR), Envelope and Spike proteins (immunohistochemistry) and anti-SARS-CoV-2 IgG and IgM antibodies (ELISA) in blood were evaluated in 10 COVID-19 donors. FINDINGS: Of 132 ocular tissues from 33 surgical-intended donors, the positivity rate for SARS-CoV-2 RNA was ~13% (17/132). Of 10 COVID-19 donors, six had PCR positive post-mortem nasopharyngeal swabs whereas eight exhibited positive post-mortem anti-SARS-CoV-2 IgG levels. Among 20 eyes recovered from 10 COVID-19 donors: three conjunctival, one anterior corneal, five posterior corneal, and three vitreous swabs tested positive for SARS-CoV-2 RNA. SARS-CoV-2 spike and envelope proteins were detected in epithelial layer of the corneas that were procured without Povidone-Iodine (PVP-I) disinfection. INTERPRETATIONS: Our study showed a small but noteworthy prevalence of SARS-CoV-2 in ocular tissues from COVID-19 donors. These findings underscore the criticality of donor screening guidelines, post-mortem nasopharyngeal PCR testing and PVP-I disinfection protocol to eliminate any tissue harboring SARS-CoV-2 being used for corneal transplantation.

Oxide rupture-induced conductivity in liquid metal nanoparticles by laser and thermal sintering.

Metallic inks with superior conductivity and printability are necessary for high-throughput manufacturing of printed electronics. In particular, gallium-based liquid metal inks have shown great potential in creating soft, flexible and stretchable electronics. Despite their metallic composition, as-printed liquid metal nanoparticle films are non-conductive due to the surrounding metal oxide shells which are primarily Ga2O3, a wide-bandgap semiconductor. Hence, these films require a sintering process to recover their conductivity. For conventional solid metallic nanoparticles, thermal and laser processing are two commonly used sintering methods, and the sintering mechanism is well understood. Nevertheless, laser sintering of liquid metal nanoparticles was only recently demonstrated, and to date, the effect of thermal sintering has rarely been investigated. Here, eutectic gallium-indium nanoparticle films are processed separately by laser or thermal sintering in an ambient environment. Laser and thermally sintered films are compared with respect to electrical conductivity, surface morphology and elemental composition, crystallinity and surface composition. Both methods impart thermal energy to the films and generate thermal stress in the particles, resulting in rupture of the gallium oxide shells and achieving electrical conductivity across the film. For laser sintering, extensive oxide rupture allows liquid metal cores to flow out and coalesce into conductive pathways. For thermal sintering, due to less thermal stress and more oxidation, the oxide shells only rupture locally and extensive phase segregation occurs, leading to non-liquid particle films at room temperature. Electrical conductivity is instead attributed to segregated metal layers and gallium oxide which becomes crystalline and conductive at high temperatures. This comprehensive comparison confirms the necessity of oxidation suppression and significant thermal stress via instantaneous laser irradiation to achieve conductive patterns in liquid form.

Transmission scanning electron microscopy: Defect observations and image simulations.

The new capabilities of a FEG scanning electron microscope (SEM) equipped with a scanning transmission electron microscopy (STEM) detector for defect characterization have been studied in parallel with transmission electron microscopy (TEM) imaging. Stacking faults and dislocations have been characterized in strontium titanate, a polycrystalline nickel-base superalloy and a single crystal cobalt-base material. Imaging modes that are similar to conventional TEM (CTEM) bright field (BF) and dark field (DF) and STEM are explored, and some of the differences due to the different accelerating voltages highlighted. Defect images have been simulated for the transmission scanning electron microscopy (TSEM) configuration using a scattering matrix formulation, and diffraction contrast in the SEM is discussed in comparison to TEM. Interference effects associated with conventional TEM, such as thickness fringes and bending contours are significantly reduced in TSEM by using a convergent probe, similar to a STEM imaging modality, enabling individual defects to be imaged clearly even in high dislocation density regions. Beyond this, TSEM provides significant advantages for high throughput and dynamic in-situ characterization.

Solute segregation and deviation from bulk thermodynamics at nanoscale crystalline defects.

It has long been known that solute segregation at crystalline defects can have profound effects on material properties. Nevertheless, quantifying the extent of solute segregation at nanoscale defects has proven challenging due to experimental limitations. A combined experimental and first-principles approach has been used to study solute segregation at extended intermetallic phases ranging from 4 to 35 atomic layers in thickness. Chemical mapping by both atom probe tomography and high-resolution scanning transmission electron microscopy demonstrates a markedly different composition for the 4-atomic-layer-thick phase, where segregation has occurred, compared to the approximately 35-atomic-layer-thick bulk phase of the same crystal structure. First-principles predictions of bulk free energies in conjunction with direct atomistic simulations of the intermetallic structure and chemistry demonstrate the breakdown of bulk thermodynamics at nanometer dimensions and highlight the importance of symmetry breaking due to the proximity of interfaces in determining equilibrium properties.

Resolved magnetic-field structure and variability near the event horizon of Sagittarius A.

Near a black hole, differential rotation of a magnetized accretion disk is thought to produce an instability that amplifies weak magnetic fields, driving accretion and outflow. These magnetic fields would naturally give rise to the observed synchrotron emission in galaxy cores and to the formation of relativistic jets, but no observations to date have been able to resolve the expected horizon-scale magnetic-field structure. We report interferometric observations at 1.3-millimeter wavelength that spatially resolve the linearly polarized emission from the Galactic Center supermassive black hole, Sagittarius A*. We have found evidence for partially ordered magnetic fields near the event horizon, on scales of ~6 Schwarzschild radii, and we have detected and localized the intrahour variability associated with these fields.

Corneal donor tissue preparation for Descemet's membrane endothelial keratoplasty.

Descemet's Membrane Endothelial Keratoplasty (DMEK) is a form of corneal transplantation in which only a single cell layer, the corneal endothelium, along with its basement membrane (Descemet's membrane) is introduced onto the recipient's posterior stroma(3). Unlike Descemet's Stripping Automated Endothelial Keratoplasty (DSAEK), where additional donor stroma is introduced, no unnatural stroma-to-stroma interface is created. As a result, the natural anatomy of the cornea is preserved as much as possible allowing for improved recovery time and visual acuity(4). Endothelial Keratoplasty (EK) is the procedure of choice for treatment of endothelial dysfunction. The advantages of EK include rapid recovery of vision, preservation of ocular integrity and minimal refractive change due to use of a small, peripheral incision(1). DSAEK utilizes donor tissue prepared with partial thickness stroma and endothelium. The rapid success and utilization of this procedure can be attributed to availability of eye-bank prepared precut tissue. The benefits of eye-bank preparation of donor tissue include elimination of need for specialized equipment in the operating room and availability of back up donor tissue in case of tissue perforation during preparation. In addition, high volume preparation of donor tissue by eye-bank technicians may provide improved quality of donor tissue. DSAEK may have limited best corrected visual acuity due to creation of a stromal interface between the donor and recipient cornea. Elimination of this interface with transplantation of only donor Descemet's membrane and endothelium in DMEK may improve visual outcomes and reduce complications after EK(5). Similar to DSAEK, long term success and acceptance of DMEK is dependent on ease of availability of precut, eye-bank prepared donor tissue. Here we present a stepwise approach to donor tissue preparation which may reduce some barriers eye-banks face in providing DMEK grafts.

Effect of microkeratome pass on tissue processing for Descemet stripping automated endothelial keratoplasty.

PURPOSE: The aim of this study was to optimize tissue preparation for Descemet stripping automated endothelial keratoplasty (DSAEK) by evaluating the outcomes of corneal tissue processing. METHODS: Forty-five corneas underwent microkeratome (MK) tissue processing for single-cut DSAEK, and 74 corneas were processed for double-cut (ultrathin) DSAEK. For single-cut processing, the corneas were cut at the thickest peripheral point (method A) or at a random location (method B). For double-cut processing, tissues were cut at the thickest peripheral point and then 180 degrees away (method C), at the thickest point and then the second thickest point (method D), or at a random peripheral starting point and then 180 degrees away (method E). The tissue was measured for corneal thickness and for endothelial cell density. RESULTS: For single-cut DSAEK tissues, there was no difference in the central tissue thickness (P = 0.23), mean peripheral thickness (P = 0.57), or peripheral tissue symmetry (P = 0.27) between A and B measured by anterior segment optical coherence tomography. For double-cut (ultrathin) DSAEK tissues, tissues cut using method C or D were not statistically significantly different for perforation rate, final central corneal thickness, mean peripheral thickness, or for tissue symmetry (P = 0.57, P = 0.33, P = 0.63, P = 0.48, respectively). All 4 tissues cut using method E were perforated during the second MK pass. The perforation, or donor loss rate, for ultrathin cut tissue preparation in group C was 23%, and for group D, it was 29%. Only 65% of successfully cut tissues in groups C and D actually achieved a thickness of ≤100 µm. CONCLUSIONS: Single-cut DSAEK tissue processing can be performed safely without peripheral corneal thickness measurements. Ultrathin DSAEK tissue processing requires peripheral thickness measurements for the first, but not for the second MK pass. Ultrathin DSAEK tissue processing led to high perforation rates. Certain tissue characteristics, processing techniques, and MK head size play a role in successful donor corneal tissue processing of ultrathin DSAEK tissue.

Evaluation of exogenous siRNA addition as a metabolic engineering tool for modifying biopharmaceuticals.

Traditional metabolic engineering approaches, including homologous recombination, zinc-finger nucleases, and short hairpin RNA, have previously been used to generate biologics with specific characteristics that improve efficacy, potency, and safety. An alternative approach is to exogenously add soluble small interfering RNA (siRNA) duplexes, formulated with a cationic lipid, directly to cells grown in shake flasks or bioreactors. This approach has the following potential advantages: no cell line development required, ability to tailor mRNA silencing by adjusting siRNA concentration, simultaneous silencing of multiple target genes, and potential temporal control of down regulation of target gene expression. In this study, we demonstrate proof of concept of the siRNA feeding approach as a metabolic engineering tool in the context of increasing monoclonal antibody (MAb) afucosylation. First, potent siRNA duplexes targeting fut8 and gmds were dosed into shake flasks with cells that express an anti-CD20 MAb. Dose response studies demonstrated the ability to titrate the silencing effect. Furthermore, siRNA addition resulted in no deleterious effects on cell growth, final protein titer, or specific productivity. In bioreactors, antibodies produced by cells following siRNA treatment exhibited improved functional characteristics compared to antibodies from untreated cells, including increased levels of afucosylation (63%), a 17-fold improvement in FCgRIIIa binding, and an increase in specific cell lysis by up to 30%, as determined in an Antibody-Dependent Cellular Cytoxicity (ADCC) assay. In addition, standard purification procedures effectively cleared the exogenously added siRNA and transfection agent. Moreover, no differences were observed when other key product quality structural attributes were compared to untreated controls. These results establish that exogenous addition of siRNA represents a potentially novel metabolic engineering tool to improve biopharmaceutical function and quality that can complement existing metabolic engineering methods.

Anterior segment optical coherence tomography versus ultrasound pachymetry to measure corneal thickness in endothelial keratoplasty donor corneas.

PURPOSE: To analyze corneal thickness (CT) by anterior segment optical coherence tomography and ultrasound pachymetry in precut endothelial keratoplasty (EK) donor corneas at the eye bank. METHODS: Thirty-six corneas were analyzed. All corneas were dissected to create lamellar grafts for EK using the standard eye bank protocol. We measured central and peripheral (3 mm from center) CT by ultrasound after lamellar tissue processing and by optical coherence tomography after the tissue was transferred to a viewing chamber. We performed paired t tests and Pearson correlation analyses to compare ultrasound and optical coherence tomography measurements. RESULTS: Central CT measurements by optical coherence tomography versus ultrasound in donor corneas were not statistically different (mean: 169 vs. 177 µm, respectively, P = 0.09). There was a strong correlation between measurements of central CT (r = 0.73, P < 0.0001). There was a statistically significant difference in mean peripheral CT measurements by optical coherence tomography and ultrasound (mean: 190 vs. 236 µm, respectively, P < 0.0001); at peripheral locations, measurements correlated moderately (r = 0.52, P = 0.002). CONCLUSIONS: Optical coherence tomography measurements of EK-prepared tissue averaged 8 µm thinner than the ultrasound measurements, similar to clinical measurements of central CT. Given the growing interest in the effect of EK graft thickness on patient outcomes, it is important to understand the differences in measurements obtained by different devices used to assess donor corneas.

Jet-launching structure resolved near the supermassive black hole in M87.

Approximately 10% of active galactic nuclei exhibit relativistic jets, which are powered by the accretion of matter onto supermassive black holes. Although the measured width profiles of such jets on large scales agree with theories of magnetic collimation, the predicted structure on accretion disk scales at the jet launch point has not been detected. We report radio interferometry observations, at a wavelength of 1.3 millimeters, of the elliptical galaxy M87 that spatially resolve the base of the jet in this source. The derived size of 5.5 ± 0.4 Schwarzschild radii is significantly smaller than the innermost edge of a retrograde accretion disk, suggesting that the M87 jet is powered by an accretion disk in a prograde orbit around a spinning black hole.

Corneal donor tissue preparation for endothelial keratoplasty.

Over the past ten years, corneal transplantation surgical techniques have undergone revolutionary changes. Since its inception, traditional full thickness corneal transplantation has been the treatment to restore sight in those limited by corneal disease. Some disadvantages to this approach include a high degree of post-operative astigmatism, lack of predictable refractive outcome, and disturbance to the ocular surface. The development of Descemet's stripping endothelial keratoplasty (DSEK), transplanting only the posterior corneal stroma, Descemet's membrane, and endothelium, has dramatically changed treatment of corneal endothelial disease. DSEK is performed through a smaller incision; this technique avoids 'open sky' surgery with its risk of hemorrhage or expulsion, decreases the incidence of postoperative wound dehiscence, reduces unpredictable refractive outcomes, and may decrease the rate of transplant rejection. Initially, cornea donor posterior lamellar dissection for DSEK was performed manually resulting in variable graft thickness and damage to the delicate corneal endothelial tissue during tissue processing. Automated lamellar dissection (Descemet's stripping automated endothelial keratoplasty, DSAEK) was developed to address these issues. Automated dissection utilizes the same technology as LASIK corneal flap creation with a mechanical microkeratome blade that helps to create uniform and thin tissue grafts for DSAEK surgery with minimal corneal endothelial cell loss in tissue processing. Eye banks have been providing full thickness corneas for surgical transplantation for many years. In 2006, eye banks began to develop methodologies for supplying precut corneal tissue for endothelial keratoplasty. With the input of corneal surgeons, eye banks have developed thorough protocols to safely and effectively prepare posterior lamellar tissue for DSAEK surgery. This can be performed preoperatively at the eye bank. Research shows no significant difference in terms of the quality of the tissue or patient outcomes using eye bank precut tissue versus surgeon-prepared tissue for DSAEK surgery. For most corneal surgeons, the availability of precut DSAEK corneal tissue saves time and money, and reduces the stress of performing the donor corneal dissection in the operating room. In part because of the ability of the eye banks to provide high quality posterior lamellar corneal in a timely manner, DSAEK has become the standard of care for surgical management of corneal endothelial disease. The procedure that we are describing is the preparation of the posterior lamellar cornea at the eye bank for transplantation in DSAEK surgery (Figure 1).