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

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

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

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

The TRPV5/6 calcium channels contain multiple calmodulin binding sites with differential binding properties

The epithelial Ca2+ channels TRPV5/6 (transient receptor potential vanilloid 5/6) are thoroughly regulated in order to fine-tune the amount of Ca2+ reabsorption. Calmodulin has been shown to be involved into calcium-dependent inactivation of TRPV5/6 channels by binding directly to the distal C-terminal fragment of the channels (de Groot et al. in Mol Cell Biol 31:2845–2853, 12). Here, we investigate this binding in detail and find significant differences between TRPV5 and TRPV6. We also identify and characterize in vitro four other CaM binding fragments of TRPV5/6, which likely are also involved in TRPV5/6 channel regulation. The five CaM binding sites display diversity in binding modes, binding stoichiometries and binding affinities, which may fine-tune the response of the channels to varying Ca2+-concentrations

High Cooperativity of the SV40 Major Capsid Protein VP1 in Virus Assembly

SV40 is a small, non enveloped DNA virus with an icosahedral capsid of 45 nm. The outer shell is composed of pentamers of the major capsid protein, VP1, linked via their flexible carboxy-terminal arms. Its morphogenesis occurs by assembly of capsomers around the viral minichromosome. However the steps leading to the formation of mature virus are poorly understood. Intermediates of the assembly reaction could not be isolated from cells infected with wt SV40. Here we have used recombinant VP1 produced in insect cells for in vitro assembly studies around supercoiled heterologous plasmid DNA carrying a reporter gene. This strategy yields infective nanoparticles, affording a simple quantitative transduction assay. We show that VP1 assembles under physiological conditions into uniform nanoparticles of the same shape, size and CsCl density as the wild type virus. The stoichiometry is one DNA molecule per capsid. VP1 deleted in the C-arm, which is unable to assemble but can bind DNA, was inactive indicating genuine assembly rather than non-specific DNA-binding. The reaction requires host enzymatic activities, consistent with the participation of chaperones, as recently shown. Our results demonstrate dramatic cooperativity of VP1, with a Hill coefficient of ∼6. These findings suggest that assembly may be a concerted reaction. We propose that concerted assembly is facilitated by simultaneous binding of multiple capsomers to a single DNA molecule, as we have recently reported, thus increasing their local concentration. Emerging principles of SV40 assembly may help understanding assembly of other complex systems. In addition, the SV40-based nanoparticles described here are potential gene therapy vectors that combine efficient gene delivery with safety and flexibility

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