Ca2+/Calmodulin Modulates TRPV1 Activation by Capsaicin

TRPV1 ion channels mediate the response to painful heat, extracellular acidosis, and capsaicin, the pungent extract from plants in the Capsicum family (hot chili peppers) (Szallasi, A., and P.M. Blumberg. 1999. Pharmacol. Rev. 51:159–212; Caterina, M.J., and D. Julius. 2001. Annu. Rev. Neurosci. 24:487–517). The convergence of these stimuli on TRPV1 channels expressed in peripheral sensory nerves underlies the common perceptual experience of pain due to hot temperatures, tissue damage and exposure to capsaicin. TRPV1 channels are nonselective cation channels (Caterina, M.J., M.A. Schumacher, M. Tominaga, T.A. Rosen, J.D. Levine, and D. Julius. 1997. Nature. 389:816–824). When activated, they produce depolarization through the influx of Na+, but their high Ca2+ permeability is also important for mediating the response to pain. In particular, Ca2+ influx is thought to be required for the desensitization to painful sensations over time (Cholewinski, A., G.M. Burgess, and S. Bevan. 1993. Neuroscience. 55:1015–1023; Koplas, P.A., R.L. Rosenberg, and G.S. Oxford. 1997. J. Neurosci. 17:3525–3537). Here we show that in inside-out excised patches from TRPV1 expressed in Xenopus oocytes and HEK 293 cells, Ca2+/calmodulin decreased the capsaicin-activated current. This inhibition was not mimicked by Mg2+, reflected a decrease in open probability, and was slowly reversible. Furthermore, increasing the calmodulin concentration in our patches by coexpression of wild-type calmodulin with TRPV1 produced inhibition by Ca2+ alone. In contrast, patches excised from cells coexpressing TRPV1 with a mutant calmodulin did not respond to Ca2+. Using an in vitro calmodulin-binding assay, we found that TRPV1 in oocyte lysates bound calmodulin, although in a Ca2+-independent manner. Experiments with GST-fusion proteins corresponding to regions of the channel NH2-terminal domain demonstrated that a stretch of ∼30 amino acids adjacent to the first ankyrin repeat bound calmodulin in a Ca2+-dependent manner. The physiological response to pain involves an influx of Ca2+ through TRPV1. Our results indicate that this Ca2+ influx may feed back on the channels, inhibiting their gating. This type of feedback inhibition could play a role in the desensitization produced by capsaicin

The genetic and environmental factors for keratoconus

Keratoconus (KC) is the most common cornea ectatic disorder. It is characterized by a cone-shaped thin cornea leading to myopia, irregular astigmatism, and vision impairment. It affects all ethnic groups and both genders. Both environmental and genetic factors may contribute to its pathogenesis. This review is to summarize the current research development in KC epidemiology and genetic etiology. Environmental factors include but are not limited to eye rubbing, atopy, sun exposure, and geography. Genetic discoveries have been reviewed with evidence from family-based linkage analysis and fine mapping in linkage region, genome-wide association studies, and candidate genes analyses. A number of genes have been discovered at a relatively rapid pace. The detailed molecular mechanism underlying KC pathogenesis will significantly advance our understanding of KC and promote the development of potential therapies

State-dependent Block of CNG Channels by Dequalinium

Cyclic nucleotide–gated (CNG) ion channels are nonselective cation channels with a high permeability for Ca(2+). Not surprisingly, they are blocked by a number of Ca(2+) channel blockers including tetracaine, pimozide, and diltiazem. We studied the effects of dequalinium, an extracellular blocker of the small conductance Ca(2+)-activated K(+) channel. We previously noted that dequalinium is a high-affinity blocker of CNGA1 channels from the intracellular side, with little or no state dependence at 0 mV. Here we examined block by dequalinium at a broad range of voltages in both CNGA1 and CNGA2 channels. We found that dequalinium block was mildly state dependent for both channels, with the affinity for closed channels 3–5 times higher than that for open channels. Mutations in the S4-S5 linker did not alter the affinity of open channels for dequalinium, but increased the affinity of closed channels by 10–20-fold. The state-specific effect of these mutations raises the question of whether/how the S4-S5 linker alters the binding of a blocker within the ion permeation pathway

Validación de los parámetros de refracción y segmento anterior mediante una nueva plataforma multi-diagnóstica (VX120)

Background: The VX120 (Visionix Luneau, France) is a novel multi-diagnostic platform that combines Hartmann–Shack based autorefraction, Placido-disk based corneal-topography and anterior segment measurements made with a stationary-Scheimpflug camera. We investigate the agreement between different parameters measured by the VX120 with accepted or gold-standard techniques to test if they are interchangeable, as well as to evaluate the repeatability and reproducibility. Methods: The right-eyes of healthy subjects were included in the study. Autorefraction of the VX120 was compared to subjective refraction. Agreement of anterior segment parameters was compared to the Sirius (CSO, Italy) including autokeratometry, central corneal thickness (CCT), iridiocorneal angle (IA). Inter and intra-test repeatability of the above parameters was assessed. Results were analyzed using Bland and Altman analyses. Results: A total of 164 eyes were evaluated. The mean difference between VX120 autorefraction and subjective refraction for sphere, spherical equivalent (SE), and cylinder was 0.01 ± 0.43 D, 0.14 ± 0.47 D, and −0.26 ± 0.30 D, respectively and high correlation was found to all parameter (r > 0.75) except for J45 (r = 0.61). The mean difference between VX120 and the Sirius system for CCT, IA, and keratometry (k1 and k2) was −3.51 ± 8.64 μm, 7.6 ± 4.2°, 0.003 ± 0.06 mm and 0.004 ± 0.04 mm, respectively and high correlation was found to all parameter (r > 0.97) except for IA (r = 0.67). Intrasession repeatability of VX120 refraction, CCT, IA and keratometry yielded low within-subject standard deviations. Inter-session repeatability showed no statistically significant difference for most of the parameters measured. Conclusions: The VX120 provides consistent refraction and most anterior segment measurements in normal healthy eyes, with high levels of intra and inter-session repeatability.Antecedentes: VX120 (Visionix Luneau, Francia) es una plataforma multi-diagnóstico novedosa que combina la auto-refracción basada en Hartmann–Shack, la topografía corneal mediante discos de Plácido, y las mediciones del segmento anterior realizadas mediante cámara de Scheimpflug. Analizamos la concordancia entre los diferentes parámetros medidos por VX120 con las técnicas aceptadas o de referencia, para probar si eran intercambiables, y evaluamos la repetibilidad y reproducibilidad. Métodos: Se incluyeron en el estudio los ojos derechos de sujetos sanos. Se comparó la auto-refracción de VX120 con la refracción subjetiva. La concordancia de los parámetros del segmento anterior se comparó con la del sistema Sirius (CSO, Italia), incluyendo autoqueratometría, espesor corneal central (ECC) y ángulo iridiocorneal (AI). Se valoró la repetibilidad inter e intra-prueba de los parámetros anteriores. Los resultados se analizaron mediante el método de Bland–Altman. Resultados: Se evaluó un total de 164 ojos. La diferencia media entre la auto-refracción de VX120 y la refracción subjetiva para esfera, equivalente esférico (EE), y cilindro fue de 0,01±0,43D, 0,14±0,47D y −0,26±0,3D, respectivamente, encontrándose una elevada correlación entre todos los parámetros (r>0,75) excepto para J45 (r=0,61). La diferencia media entre VX120 y el sistema Sirius para ECC, AI, y queratometría (k1 y k2) fue de -3,51±8,64 μm, 7,6±4,2°, 0,003±0,06 mm y 0,004±0,04 mm, respectivamente, encontrándose una elevada correlación entre todos los parámetros (r>0,97) excepto para AI (r=0,67). La repetibilidad intra-sesión de la refracción VX120, ECC, AI y queratometría reflejó desviaciones estándar bajas entre sujetos. La repetibilidad inter-sesión no reflejó una diferencia significativa para la mayoría de los parámetros medidos. Conclusiones: VX120 aporta medidas consistentes de refracción y de la mayoría de las mediciones del segmento anterior en ojos sanos normales, con elevados niveles de repetibilidad intra e inter-sesión

IMI : global trends in myopia management attitudes and strategies in clinical practice : 2022 update

PURPOSE. Surveys in 2015 and 2019 identified a high level of eye care practitioner concern/activity about myopia, but the majority still prescribed single vision interventions to young myopes. This research aimed to provide updated information. METHODS. A self-administered, internet-based questionnaire was distributed in 13 languages, through professional bodies to eye care practitioners globally. The questions examined awareness of increasing myopia prevalence, perceived efficacy and adoption of available strategies, and reasons for not adopting specific strategies. RESULTS. Of the 3195 respondents, practitioners’ concern about the increasing frequency of pediatric myopia in their practices differed between continents (P < 0.001), being significantly higher in Asia (9.0 ± 1.5 of 10) than other continents (range 7.7–8.2; P ≤ 0.001). Overall, combination therapy was perceived by practitioners to be the most effective method of myopia control, followed by orthokeratology and pharmaceutical approaches. The least effective perceived methods were single vision distance undercorrection, spectacles and contact lenses, as well as bifocal spectacles. Practitioners rated their activity in myopia control between (6.6 ± 2.9 in South America to 7.9 ± 1.2/2.2 in Australasia and Asia). Single-vision spectacles are still the most prescribed option for progressing young myopia (32.2%), but this has decreased since 2019, and myopia control spectacles (15.2%), myopia control contact lenses (8.7%) and combination therapy (4.0%) are growing in popularity. CONCLUSIONS. More practitioners across the globe are practicing myopia control, but there are still significant differences between and within continents. Practitioners reported that embracing myopia control enhanced patient loyalty, increasing practice revenue and improving job satisfaction

Validation of keratometric measurements obtained with a new integrated aberrometry-topography system

Purpose: A clinical evaluation of the L80 videokeratographer (Visionix Luneau, Chartres, France) was performed to assess its validity and repeatability compared with a traditional Bausch and Lomb (B & L) keratometer. Methods: 87 right eyes of 87 subjects, (mean age 23.72 ± 3.62 years old, 70 women and 17 men), participated in this study. Corneal curvature was measured using the L80 instrument by one practitioner and the manual B & L keratometer by a different practitioner. Intratest and intertest repeatability were assessed. Results: Corneal curvature was found to be statistically different between the two instruments (p < 0.001), with the L80 providing a slightly steeper bias of 0.05 mm and 0.07 mm for the horizontal and vertical meridians, respectively than the B & L keratometer. 78.2% and 86.2% of the L80 results were within ±0.1 mm (±0.06 D) and 95.4% and 97.7% within ±0.2 mm (±0.11 D) of the readings obtained with the B & L keratometer along the horizontal and the vertical meridians, respectively. The agreement between the L80 and B & L keratometers axes was 31.0% within ±5°, 54.0% within ±10°, 60.9% within ±15°, 71.3% within ±20° and 87.4% within ±40°. Intratest repeatability was the same for both instruments. Intertest repeatability was better for the L80 videokeratographer compared to the B & L keratometer and showed no significant difference between the two sessions. Conclusion: The L80 videokeratographer is a reliable objective instrument comparable to other autokeratometers which, in addition, combines many other useful clinical features. It provides steeper radii of curvature measurements than the B & L keratometer. An offset incorporated into the instrument could mitigate the difference between the two instruments and make them interchangeable

Bilateral chorioretinal coloboma discovered with ultra-wide field retinal imaging

Uveal coloboma results from incomplete closure of the optic cup fissure. While conducting an evaluation of a new ultra-wide field retinal imaging camera (Optomap), which provides a view of the fundus up to 200° at one time without mydriasis, we discovered a case of bilateral chorioretinal coloboma in a 21-year-old student. The lesion was located in the midperiphery of each eye less than 2 disc diameters (DD) below the optic disc in the inferonasal quadrants. The size of the coloboma in the right eye was 1.8 DD in height and 1.3 DD in width, while the left lesion was 2.4 DD in height and 2.6 DD in width. The subject was totally asymptomatic and without any complication such as retinal detachment or choroidal neovascularization, which often accompany this type of lesion. The visual field of each eye displayed an absolute scotoma corresponding to the size and location of the coloboma. No management was necessary but the subject was advised to report for visual examination at regular intervals because complications can occur at any age

The Abundant Nuclear Enzyme PARP Participates in the Life Cycle of Simian Virus 40 and Is Stimulated by Minor Capsid Protein VP3

The abundant nuclear enzyme poly(ADP-ribose) polymerase (PARP) functions in DNA damage surveillance and repair and at the decision between apoptosis and necrosis. Here we show that PARP binds to simian virus 40 (SV40) capsid proteins VP1 and VP3. Furthermore, its enzymatic activity is stimulated by VP3 but not by VP1. Experiments with purified mutant proteins demonstrated that the PARP binding domain in VP3 is localized to the 35 carboxy-terminal amino acids, while a larger peptide of 49 amino acids was required for full stimulation of its activity. The addition of 3-aminobenzamide (3-AB), a known competitive inhibitor of PARP, demonstrated that PARP participates in the SV40 life cycle. The titer of SV40 propagated on CV-1 cells was reduced by 3-AB in a dose-dependent manner. Additional experiments showed that 3-AB did not affect viral DNA replication or capsid protein production. PARP did not modify the viral capsid proteins in in vitro poly(ADP-ribosylation) assays, implying that it does not affect SV40 infectivity. On the other hand, it greatly reduced the magnitude of the host cytopathic effects, a hallmark of SV40 infection. Additional experiments suggested that the stimulation of PARP activity by VP3 leads the infected cell to a necrotic pathway, characterized by the loss of membrane integrity, thus facilitating the release of mature SV40 virions from the cells. Our studies identified a novel function of the minor capsid protein VP3 in the recruitment of PARP for the SV40 lytic process

Cellular Transcription Factor Sp1 Recruits Simian Virus 40 Capsid Proteins to the Viral Packaging Signal, ses

Simian virus 40 (SV40) capsid assembly occurs in the nucleus. All three capsid proteins bind DNA nonspecifically, raising the dilemma of how they attain specificity to the SV40 minichromosome in the presence of a large excess of genomic DNA. The SV40 packaging signal, ses, which is required for assembly, is composed of multiple DNA elements that bind transcription factor Sp1. Our previous studies showed that Sp1 participates in SV40 assembly and that it cooperates in DNA binding with VP2/3. We hypothesized that Sp1 recruits the capsid proteins to the viral minichromosome, conferring upon them specific DNA recognition. Here, we have tested the hypothesis. Computer analysis showed that the combination of six tandem GC boxes at ses is not found at cellular promoters and therefore is unique to SV40. Cooperativity in DNA binding between Sp1 and VP2/3 was not abolished at even a 1,000-fold excess of cellular DNA, providing strong support for the recruitment hypothesis. Sp1 also binds VP1 and cooperates with VP1 in DNA binding. VP1 pentamers (VP1(5)) avidly interact with VP2/3, utilizing the same VP2/3 domain as described for polyomavirus. We conclude that VP1(5)-VP2/3 building blocks are recruited by Sp1 to ses, where they form the nucleation center for capsid assembly. By this mechanism the virus ensures that capsid formation is initiated at a single site around its minichromosome. Sp1 enhances the formation of SV40 pseudovirions in vitro, providing additional support for the model. Analyses of Sp1 and VP3 deletion mutants showed that Sp1 and VP2/3 bind one another and cooperate in DNA binding through their DNA-binding domains, with additional contacts outside these domains. VP1 contacts Sp1 at residues outside the Sp1 DNA-binding domain. These and additional data allowed us to propose a molecular model for the VP1(5)-VP2/3-DNA-Sp1 complex