Cecinio Ronquillo

Promoter polymorphism of the erythropoietin gene in severe diabetic eye and kidney complications

Significant morbidity and mortality among patients with diabetes mellitus result largely from a greatly increased incidence of microvascular complications. Proliferative diabetic retinopathy (PDR) and end stage renal disease (ESRD) are two of the most common and severe microvascular complications of diabetes. A high concordance exists in the development of PDR and ESRD in diabetic patients, as well as strong familial aggregation of these complications, suggesting a common underlying genetic mechanism. However, the precise gene(s) and genetic variant(s) involved remain largely unknown. Erythropoietin (EPO) is a potent angiogenic factor observed in the diabetic human and mouse eye. By a combination of case–control association and functional studies, we demonstrate that the T allele of SNP rs1617640 in the promoter of the EPO gene is significantly associated with PDR and ESRD in three European-American cohorts [Utah: P = 1.91 × 10−3; Genetics of Kidneys in Diabetes (GoKinD) Study: P = 2.66 × 10−8; and Boston: P = 2.1 × 10−2]. The EPO concentration in human vitreous body was 7.5-fold higher in normal subjects with the TT risk genotype than in those with the GG genotype. Computational analysis suggests that the risk allele (T) of rs1617640 creates a matrix match with the EVI1/MEL1 or AP1 binding site, accounting for an observed 25-fold enhancement of luciferase reporter expression as compared with the G allele. These results suggest that rs1617640 in the EPO promoter is significantly associated with PDR and ESRD. This study identifies a disease risk-associated gene and potential pathway mediating severe diabetic microvascular complications.

Senior-Loken syndrome: a syndromic form of retinal dystrophy associated with nephronophthisis.

Senior-Løken syndrome (SLS) is an autosomal recessive disease characterized by development of a retinitis pigmentosa (RP)- or Leber congenital amaurosis (LCA)-like retinal dystrophy and a medullary cystic kidney disease, nephronophthisis. Mutations in several genes (called nephrocystins) have been shown to cause SLS. The proteins encoded by these genes are localized in the connecting cilium of photoreceptor cells and in the primary cilium of kidney cells. Nephrocystins are thought to have a role in regulating transport of proteins bound to the outer segment/primary cilium; however, the precise molecular mechanisms are largely undetermined. This review will survey the biochemistry, cell biology and existing animal models for each of the nephrocystins as it relates to photoreceptor biology and pathogenesis of retinal degeneration.

Optimum on-time duty cycle for micropulse technology

Purpose To evaluate the optimum on time for the most efficient removal of lens fragments using micropulsed ultrasound (US). Setting John A. Moran Eye Center Laboratories, University of Utah, Salt Lake City, Utah, USA. Design Experimental study. Methods Twenty porcine lens nuclei were soaked in formalin for 2 hours and then divided into 2.0 mm cubes. Using an US machine with a 0.9 mm bent and a 30‐degree bevel tip, the on time was varied every millisecond (ms) from 2 ms to 10 ms with the off time kept constant at 10 ms. Efficiency (time to lens removal) and chatter (number of lens fragment repulsions from the tip) were determined. Results The most efficient phacoemulsification was achieved with an on time of 6 ms. On times shorter than 6 ms were significantly less efficient (R2 = .82, P=.04). Greater on times did not result in a significant difference in efficiency (R2 = .03, P=.78) but did appear to have more chatter events when comparing 9 to 10 ms with 2 to 8 ms (P<.0001). Conclusions With micropulsed longitudinal US, a 6 ms on time was equally as efficient as longer on times, while shorter on times (2 to 5 ms) had decreased efficiency. At 9 ms and 10 ms on time, significantly more chatter was noted. Therefore, to maximize phacoemulsification, an on‐time setting of 6 ms is recommended. Financial Disclosure No author has a financial or proprietary interest in any material or method mentioned.

Heterotrimeric Kinesin-2 (KIF3) Mediates Transition Zone and Axoneme Formation of Mouse Photoreceptors

Background: Heterotrimeric kinesin-2 (KIF3) has been implicated in intraflagellar trafficking of photoreceptor membrane proteins by IFT. Results: KIF3 and IFT88 are required for transition zone and axoneme formation, but are dispensable for rhodopsin trafficking. Conclusion: Transmembrane proteins, including rhodopsin, traffic to the OS even when IFT is disabled. Significance: KIF3 builds and maintains the photoreceptor transition zone and axoneme that are essential for photoreceptor integrity and vision. Anterograde intraflagellar transport (IFT) employing kinesin-2 molecular motors has been implicated in trafficking of photoreceptor outer segment proteins. We generated embryonic retina-specific (prefix “emb”) and adult tamoxifen-induced (prefix “tam”) deletions of KIF3a and IFT88 in adult mice to study photoreceptor ciliogenesis and protein trafficking. In embKif3a−/− and in embIft88−/− mice, basal bodies failed to extend transition zones (connecting cilia) with outer segments, and visual pigments mistrafficked. In contrast, tamKif3a−/− and tamIft88−/− photoreceptor axonemes disintegrated slowly post-induction, starting distally, but rhodopsin and cone pigments trafficked normally for more than 2 weeks, a time interval during which the outer segment is completely renewed. The results demonstrate that visual pigments transport to the retinal outer segment despite removal of KIF3 and IFT88, and KIF3-mediated anterograde IFT is responsible for photoreceptor transition zone and axoneme formation.

Determining optimal ultrasound off time with micropulse longitudinal phacoemulsification

Purpose To evaluate the optimum off time for the most efficient removal of lens fragments using micropulse ultrasound (US). Setting John A. Moran Eye Center Laboratories, University of Utah, Salt Lake City, Utah, USA. Design Experimental study. Methods Porcine lens nuclei were soaked in formalin for 2 hours and then cut into 2.0 mm cubes using the Signature US machine with a bent 0.9 mm phaco tip with a 30‐degree bevel. The on time was 7 milliseconds (ms), and the off time was varied from 2 to 20 ms in 2 ms steps. Phacoemulsification efficiency (time for fragment removal) and chatter (number of times the fragment bounced from the tip) were measured. Results A nonsignificant linear increase in efficiency was observed with 2 to 6 ms of off time (R2 = .87, P = .24). A significant linear decrease in efficiency was observed with 6 to 20 ms (R2 = .74, P = .006). Conclusions With micropulse longitudinal US, 6 to 7 ms of off time was as efficient as shorter off times; longer off times (8 to 20 ms) showed decreased efficiency. Chatter was minimal and statistically similar throughout. To maximize phacoemulsification US efficiency, an off‐time setting of 6 ms is recommended. Financial Disclosure No author has a financial or proprietary interest in any material or method mentioned.

Effect of phaco tip diameter on efficiency and chatter.

Purpose To evaluate 3 phacoemulsification tips of different sizes and determine which size is most efficient in lens fragment removal using 3 ultrasound (US) approaches. Setting John A. Moran Eye Center Laboratories, University of Utah, Salt Lake City, Utah, USA. Design Experimental study. Methods Porcine lens nuclei were formalin‐soaked for 2 hours then divided into 2.0 mm cubes; 1.1 mm, 0.9 mm, and 0.7 mm phaco tips were used with torsional and micropulsed US. The 1.1 mm tips were unavailable for torsional US, so 0.9 mm and 0.7 mm tips were used. Efficiency (amount of time for lens removal) and chatter (number of lens‐fragment repulsions from the tip) were determined. Results The mean phacoemulsification efficiency was highest with the 0.9 mm tip for all US variations. There were statistically significant differences between the 0.9 mm and 0.7 mm tips with micropulsed US (0.8 seconds ± 0.29 [SD] versus 1.4 ± 0.93 seconds; P=.0112) and transversal US (0.8 ± 0.17 seconds versus 1.4 ± 0.89 seconds; P=.0065). There was no significant difference between 0.9 mm and 0.7 mm tips with torsional US or between the 1.1 mm and 0.9 mm tips with micropulsed or transversal US; however, trends were identical, with 0.9 mm tips performing better than 0.7 mm and 1.1 mm tips. Conclusion With all 3 systems, the 0.9 mm tip was most efficient, with the fewest outliers and smallest standard deviation. Financial Disclosure No author has a financial or proprietary interest in any material or method mentioned.

Determining Optimal Torsional Ultrasound Power for Cataract Surgery With Automatic Longitudinal Pulses at Maximum Vacuum Ex Vivo

PURPOSE To determine the optimal longitudinal power settings for Infiniti OZil Intelligent Phaco (IP) at varying torsional amplitude settings; and to test the hypothesis that increasing longitudinal power is more important at lower torsional amplitudes to achieve efficient phacoemulsification. DESIGN Laboratory investigation. METHODS setting: John A. Moran Eye Center, University of Utah, Salt Lake City, Utah. procedure: Individual porcine nuclei were fixed in formalin, then cut into 2.0 mm cubes. Lens cube phacoemulsification was done using OZil IP at 60%, 80%, and 100% torsional amplitude with 0%, 10%, 20%, 30%, 50%, 75%, or 100% longitudinal power. All experiments were done using a 20 gauge 0.9 mm bent reverse bevel phaco tip at constant vacuum (550 mm Hg), aspiration rate (40 mL/min), and bottle height (50 cm). main outcome measure: Complete lens particle phacoemulsification (efficiency). RESULTS Linear regression analysis showed a significant increase in efficiency with increasing longitudinal power at 60% torsional amplitude (R(2) = 0.7269, P = .01) and 80% torsional amplitude (R(2) = 0.6995, P = .02) but not at 100% amplitude (R(2) = 0.3053, P = .2). Baseline comparison of 60% or 80% vs 100% torsional amplitude without longitudinal power showed increased efficiency at 100% (P = .0004). Increasing longitudinal power to 20% abolished the efficiency difference between 80% vs 100% amplitudes. In contrast, 75% longitudinal power abolished the efficiency difference between 60% vs 100% torsional amplitudes. CONCLUSIONS Results suggest that longitudinal power becomes more critical at increasing phacoemulsification efficiencies at torsional amplitudes less than 100%. Increasing longitudinal power does not further increase efficiency at maximal torsional amplitudes.

Ciliopathy-associated IQCB1/NPHP5 protein is required for mouse photoreceptor outer segment formation

Null mutations in the human IQCB1/NPHP5 (nephrocystin‐5) gene that encodes NPHP5 are the most frequent cause of Senior‐Løken syndrome, a ciliopathy that is characterized by Leber congenital amaurosis and nephronophthisis. We generated germline Nphp5‐knockout mice by placing a b‐Geo gene trap in intron 4, thereby truncating NPHP5 at Leu87 and removing all known functional domains. At eye opening, Nphp5‐/‐ mice exhibited absence of scotopic and photopic electroretinogram responses, a phenotype that resembles Leber congenital amaurosis. Outer segment transmembrane protein accumulation in Nphp5‐/‐ endoplasmic reticulum was evident as early as postnatal day (P)6. EGFP‐CETN2, a centrosome and transition zone marker, identified basal bodies in Nphp5‐/‐ photoreceptors, but without fully developed transition zones. Ultrastructure of P6 and 10 Nphp5‐/‐ photoreceptors revealed aberrant transition zones of reduced diameter. Nphp5‐/‐ photoreceptor degeneration was complete at 1 mo of age but was delayed significantly in Nphp5‐/‐;Nrl‐/‐ (cone only) retina. Nphp5‐/‐ mouse embryonic fibroblast developed normal cilia, and Nphp5‐/‐ kidney histology at 1 yr of age showed no significant pathology. Results establish that nephrocystin‐5 is essential for photoreceptor outer segment formation but is dispensable for kidney and mouse embryonic fibroblast ciliary formation.—Ronquillo, C. C., Hanke‐Gogokhia, C., Revelo, M. P., Frederick, J. M., Jiang, L., Baehr, W. Ciliopathy‐associated IQCB1/NPHP5 protein is required for mouse photoreceptor outer segment formation. FASEB J. 30, 3400–3412 (2016). www.fasebj.org

Bent versus straight tips in micropulsed longitudinal phacoemulsification

OBJECTIVE The aim of this study was to evaluate bent and straight phacoemulsification tips to determine which tip is more efficient in removal of lens fragments, using micropulsed longitudinal ultrasound in phacoemulsification. DESIGN In vitro laboratory study. METHODS The John A. Moran Eye Center Laboratories, University of Utah, Salt Lake City, Utah, was the study setting. Pig lenses hardened in a manner comparable with dense human cataracts were cut into 2-mm cubes and removed with micropulsed longitudinal ultrasound using settings previously shown to be optimally efficient (6 milliseconds on and 6 milliseconds off for a bent tip). To verify this time as most efficient for a straight tip, we also tested times of 5, 6, and 7 milliseconds time on and off. The tips were either straight or with a 20-degree bend. Twenty cubes were used for each comparative run. RESULTS For the straight tip, 6 milliseconds on (1.56 ± 0.815 seconds) was significantly more efficient than 7 milliseconds on (2.45 ± 1.56 seconds, p = 0.001) and not significantly more efficient than 5 milliseconds on (1.69 ± 0.86 seconds, p = 0.43). Five milliseconds off time (1.45 ± 0.76s) was more efficient than 6 milliseconds (2.06 ± 1.37 seconds, p = 0.004) and 7 milliseconds off (2.18 ± 1.24s, p = 0.001). The straight tip was more efficient than the bent tip (1.38 ± 0.83 versus 2.93 ± 2.14 seconds, p = 0.006). CONCLUSIONS Results are contrary to accepted common belief. Micropulsed longitudinal phacoemulsification is more efficient with a straight rather than a bent tip.

The impact of tip bevel angulation on phacoemulsification efficiency and chatter

Primary objective: To evaluate the effect of tip bevel angulation on phacoemulsification efficiency and chatter. Research design: In vitro laboratory study. Methods and procedures: Formalin-soaked porcine lenses were divided into 2 mm cubes. 0.9 mm straight 0, 15, 30, 45 beveled degree tips were used with micropulse ultrasound (6 ms on and 6 ms off ). Power was set at 100%, vacuum levels were set at 500 mmHg; and aspiration rates were set at 50 mL/min. Efficiency (time to lens removal) and chatter (number of lens fragment repulsions from the tip) were determined. Main outcomes and results: Changing the bevel angulation on a straight 0.9 mm phacoemulsification tip had no significant effect on efficiency. A 45 degree bevel was the most efficient tip overall. Chatter was seen to be significantly higher with a 15 degree tip (ANOVA, P=.0046). Conclusions: Tip bevel angulation has little effect on phacoemulsification efficiency and chatter, especially when optimized parameters are used. Limitations of this study include use of only one ultrasound power modulation and hard nuclear material.