Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 29
Filtrar
1.
Trials ; 25(1): 384, 2024 Jun 14.
Artículo en Inglés | MEDLINE | ID: mdl-38877566

RESUMEN

BACKGROUND: In recent years, alternative monitoring approaches, such as risk-based and remote monitoring techniques, have been recommended instead of traditional on-site monitoring to achieve more efficient monitoring. Remote risk-based monitoring (R2BM) is a monitoring technique that combines risk-based and remote monitoring and focuses on the detection of critical data and process errors. Direct data capture (DDC), which directly collects electronic source data, can facilitate R2BM by minimizing the extent of source documents that must be reviewed and reducing the additional workload on R2BM. In this study, we evaluated the effectiveness of R2BM and the synergistic effect of combining R2BM with DDC. METHODS: R2BM was prospectively conducted with eight participants in a randomized clinical trial using a remote monitoring system that uploaded photographs of source documents to a cloud location. Critical data and processes were verified by R2BM, and later, all were confirmed by on-site monitoring to evaluate the ability of R2BM to detect critical data and process errors and the workload of uploading photographs for clinical trial staff. In addition, the reduction of the number of uploaded photographs was evaluated by assuming that the DDC was introduced for data collection. RESULTS: Of the 4645 data points, 20.9% (n = 973, 95% confidence interval = 19.8-22.2) were identified as critical. All critical data errors corresponding to 5.4% (n = 53/973, 95% confidence interval = 4.1-7.1) of the critical data and critical process errors were detectable by R2BM. The mean number of uploaded photographs and the mean time to upload them per visit per participant were 34.4 ± 11.9 and 26.5 ± 11.8 min (mean ± standard deviation), respectively. When assuming that DDC was introduced for data collection, 45.0% (95% confidence interval = 42.2-47.9) of uploaded photographs for R2BM were reduced. CONCLUSIONS: R2BM can detect 100% of the critical data and process errors without on-site monitoring. Combining R2BM with DDC reduces the workload of R2BM and further improves its efficiency.


Asunto(s)
Fotograbar , Humanos , Estudios Prospectivos , Medición de Riesgo , Carga de Trabajo , Nube Computacional , Recolección de Datos/métodos , Femenino , Masculino , Exactitud de los Datos , Proyectos de Investigación
2.
Sci Rep ; 14(1): 1749, 2024 01 19.
Artículo en Inglés | MEDLINE | ID: mdl-38242937

RESUMEN

Optogenetics enables precise regulation of intracellular signaling in target cells. However, the application of optogenetics to induce the differentiation of precursor cells and generate mature cells with specific functions has not yet been fully explored. Here, we focused on osteoclasts, which play an important role in bone remodeling, to develop a novel optogenetics tool, Opto-RANK, which can manipulate intracellular signals involved in osteoclast differentiation and maturation using blue light. We engineered Opto-RANK variants, Opto-RANKc and Opto-RANKm, and generated stable cell lines through retroviral transduction. Differentiation was induced by blue light, and various assays were conducted for functional analysis. Osteoclast precursor cells expressing Opto-RANK differentiated into multinucleated giant cells on light exposure and displayed upregulation of genes normally induced in differentiated osteoclasts. Furthermore, the differentiated cells exhibited bone-resorbing activities, with the possibility of spatial control of the resorption by targeted light illumination. These results suggested that Opto-RANK cells differentiated by light possess the features of osteoclasts, both morphological and functional. Thus, Opto-RANK should be useful for detailed spatiotemporal analysis of intracellular signaling during osteoclast differentiation and the development of new therapies for various bone diseases.


Asunto(s)
Resorción Ósea , Osteoclastos , Humanos , Osteoclastos/metabolismo , Resorción Ósea/metabolismo , Luz Azul , Optogenética , Diferenciación Celular/genética , Ligando RANK/metabolismo
3.
Biochem Biophys Res Commun ; 690: 149231, 2024 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-38000293

RESUMEN

Cell fusion plays a key role in the development and formation of tissues and organs in several organisms. Skeletal myogenesis is assessed in vitro by cell shape and gene and protein expression using immunofluorescence and immunoblotting assays. However, these conventional methods are complex and do not allow for easy time-course observation in living cells. Therefore, this study aimed to develop a Cre recombination-based fluorescent reporter system to monitor cell-cell fusion. We combined green and red fluorescent proteins with a Cre-loxP system to detect syncytium formation using a fluorescent binary switch. This allowed us to visualize mononucleated cells with green fluorescence before fusion and multinucleated syncytia with red fluorescence by conditional expression after cell fusion. The formation of multinuclear myotubes during myogenic differentiation was detected by the change in fluorescence from green to red after Cre-mediated recombination. The distribution of the fluorescence signal correlated with the expression of myogenic differentiation markers. Moreover, red reporter fluorescence intensity was correlated with the number of nuclei contained in the red fluorescent-positive myotubes. We also successfully demonstrated that our fusion monitoring system is applicable to the formation of skeletal muscle myotube and placental syncytiotrophoblast. These results suggest that the color-switching fluorescent reporter system, using Cre-mediated recombination, could be a robust tool used to facilitate the study of cell-to-cell fusion.


Asunto(s)
Placenta , Proteína Fluorescente Roja , Embarazo , Femenino , Humanos , Fusión Celular , Placenta/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Diferenciación Celular/genética , Recombinación Genética , Integrasas/genética , Integrasas/metabolismo , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo
4.
Chem Pharm Bull (Tokyo) ; 71(10): 792-797, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37779082

RESUMEN

Chiral lithium binaphtholates prepared from the corresponding binaphthols and lithium tert-butoxide effectively catalyze the asymmetric Michael additions of ketones to poorly reactive acrylamides. The lithium binaphtholate catalyst mediates ketone deprotonation and enantioselective carbon-carbon bond formation to the acrylamide to deliver the Michael adduct in good yield and enantioselectivity. A small excess of lithium tert-butoxide relative to the binaphthol successfully enolizes the ketone in the initial stage of the reaction to promote the Michael reaction. Computational analysis of the transition state suggested that the 3- and 3'-phenyl groups of the binaphtholate catalyst regulate the orientation of the lithium enolate and the subsequent approach of the acrylamide, leading to superior enantioselectivity.


Asunto(s)
Acrilamidas , Litio , Litio/química , Acrilamida , Estereoisomerismo , Cetonas/química , Catálisis
6.
Phytochemistry ; 206: 113548, 2023 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-36481317

RESUMEN

Antiosteoclastogenic-guided screening was conducted with 120 extracts of the medicinal plants collected in Egypt that led to the selection of Artemisia judaica L. (Asteraceae). Three undescribed davanone-related terpenoids, arteperoxides A-C, were isolated from the extract with two known derivatives, hydroxydavanone and davana acid. Structural analysis revealed that arteperoxides A-C were tris-normonoterpene-sesquiterpene conjugates with peroxide bridges. Although davanone derivatives with peroxides, such as a hydroperoxyl and peroxyhemiketal groups, have been isolated from Artemisia species, arteperoxides A-C are the first variations observed to contain peroxide bridges between two terpene-derived units. The absolute configurations of arteperoxides A and B were studied based on their spectroscopic data compared with those of the semisynthetic analogs that have ether linkages. The natural and synthetic compounds were tested for the antiosteoclastogenic activity, and arteperoxide C and hydroxydavanone were more potent than other compounds at 20 µM.


Asunto(s)
Artemisia , Plantas Medicinales , Sesquiterpenos , Artemisia/química , Peróxidos , Sesquiterpenos/farmacología , Sesquiterpenos/química , Terpenos , Extractos Vegetales/farmacología , Extractos Vegetales/química
7.
J Med Chem ; 65(4): 3460-3472, 2022 02 24.
Artículo en Inglés | MEDLINE | ID: mdl-35113551

RESUMEN

Three new diterpenes, stellejasmins A (1) and B (2) and 12-O-benzoylphorbol-13-heptanoate (3), were isolated from the roots of Stellera chamaejasme L. The structures of 1-3 were elucidated by extensive NMR and mass spectroscopic analyses. Compounds 1 and 2 are the first derivatives containing a hydroxy group at C-2 in the family of daphnane and tigliane diterpenes. The presence of a chlorine atom in 1 is unique in the plant metabolite. Compound 3 has an odd-number acyl group, which is biosynthetically notable. Human immunodeficiency virus (HIV) LTR-driven transcription activity was tested with 1-3 and 17 known diterpenes isolated from S. chamaejasme L. and Wikstroemia retusa A.Gray. Among these, gnidimacrin (4), stelleralide A (5), and wikstroelide A (20) were highly potent, with EC50 values of 0.14, 0.33, and 0.39 nM, respectively. The structure-activity relationship (SAR) was investigated using 20 natural and eight synthetic diterpenes. This is the first SAR study on natural daphnane and tigliane diterpenes.


Asunto(s)
Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/farmacología , Diterpenos/síntesis química , Diterpenos/farmacología , VIH/efectos de los fármacos , Forboles/química , Latencia del Virus/efectos de los fármacos , Diterpenos/química , Modelos Moleculares , Simulación del Acoplamiento Molecular , Forboles/farmacología , Extractos Vegetales/química , Extractos Vegetales/farmacología , Raíces de Plantas/química , Relación Estructura-Actividad , Thymelaeaceae/química , Wikstroemia/química
8.
Cell Rep Med ; 2(6): 100298, 2021 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-34195678

RESUMEN

Duchenne muscular dystrophy (DMD) is a muscle degenerating disease caused by dystrophin deficiency, for which therapeutic options are limited. To facilitate drug development, it is desirable to develop in vitro disease models that enable the evaluation of DMD declines in contractile performance. Here, we show MYOD1-induced differentiation of hiPSCs into functional skeletal myotubes in vitro with collagen gel and electrical field stimulation (EFS). Long-term EFS training (0.5 Hz, 20 V, 2 ms, continuous for 2 weeks) mimicking muscle overuse recapitulates declines in contractile performance in dystrophic myotubes. A screening of clinically relevant drugs using this model detects three compounds that ameliorate this decline. Furthermore, we validate the feasibility of adapting the model to a 96-well culture system using optogenetic technology for large-scale screening. Our results support a disease model using patient-derived iPSCs that allows for the recapitulation of the contractile pathogenesis of DMD and a screening strategy for drug development.


Asunto(s)
Distrofina/genética , Estimulación Eléctrica/métodos , Células Madre Pluripotentes Inducidas/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular de Duchenne/genética , Compuestos de Boro/farmacología , Sistemas CRISPR-Cas , Diferenciación Celular , Colágeno/química , Creatina/farmacología , Dantroleno/farmacología , Distrofina/deficiencia , Geles , Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/citología , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Modelos Biológicos , Contracción Muscular/efectos de los fármacos , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/efectos de los fármacos , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patología , Optogenética , Cultivo Primario de Células , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo
9.
Adv Exp Med Biol ; 1293: 359-375, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33398826

RESUMEN

This chapter describes the current progress of basic research, and potential therapeutic applications primarily focused on the optical manipulation of muscle cells and neural stem cells using microbial rhodopsin as a light-sensitive molecule. Since the contractions of skeletal, cardiac, and smooth muscle cells are mainly regulated through their membrane potential, several studies have been demonstrated to up- or downregulate the muscle contraction directly or indirectly using optogenetic actuators or silencers with defined stimulation patterns and intensities. Light-dependent oscillation of membrane potential also facilitates the maturation of myocytes with the development of T tubules and sarcomere structures, tandem arrays of minimum contractile units consists of contractile proteins and cytoskeletal proteins. Optogenetics has been applied to various stem cells and multipotent/pluripotent cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to generate light-sensitive neurons and to facilitate neuroscience. The chronic optical stimulation of the channelrhodopsin-expressing neural stem cells facilitates their neural differentiation. There are potential therapeutic applications of optogenetics in cardiac pacemaking, muscle regeneration/maintenance, locomotion recovery for the treatment of muscle paralysis due to motor neuron diseases such as amyotrophic lateral sclerosis (ALS). Optogenetics would also facilitate maturation, network integration of grafted neurons, and improve the microenvironment around them when applied to stem cells.


Asunto(s)
Células Madre Pluripotentes Inducidas , Células-Madre Neurales , Células Musculares , Neuronas , Optogenética
10.
Int Ophthalmol ; 41(1): 151-162, 2021 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-32894391

RESUMEN

PURPOSE: This study searched for early predictive vascular biomarkers for visual outcomes in eyes with macular edema caused by branch retinal vein occlusion (BRVOME). METHODS: Twenty-four eyes of 24 subjects with BRVOME were treated with the intravitreal injection of ranibizumab (IVR) for at least 6 months. We measured mean blur rate (MBR) in the optic nerve head (ONH) and vessel density (VD) in the macula with laser speckle flowgraphy and optical coherence tomography angiography, respectively. RESULTS: Six-month post-IVR best-corrected visual acuity (BCVA) was correlated positively with age, pre-IVR BCVA, 1-month post-IVR BCVA, 3-month post-IVR BCVA and pre-IVR systolic blood pressure (P < 0.001, P < 0.001, P < 0.001, P < 0.001 and P = 0.02, respectively) and negatively with pre-IVR overall MBR, 1-month post-IVR overall MBR, 6-month post-IVR overall MBR, 3-month post-IVR deep retinal capillary plexus (DCP) VD and 6-month post-IVR DCP VD (P = 0.03, P = 0.03, P = 0.02, P = 0.01 and P = 0.005, respectively). Furthermore, a multiple regression analysis showed that pre-IVR overall MBR (ß = - 0.67, P = 0.009) was among independent prognostic factors predicting 6-month post-IVR BCVA. Six-month post-IVR DCP VD was also correlated with overall MBR at all time points. CONCLUSION: ONH blood flow may be a pre-IVR biomarker of both visual outcomes and post-IVR deep macular microcirculation in eyes with BRVOME.


Asunto(s)
Edema Macular , Oclusión de la Vena Retiniana , Inhibidores de la Angiogénesis/uso terapéutico , Angiografía con Fluoresceína , Humanos , Inyecciones Intravítreas , Rayos Láser , Edema Macular/diagnóstico , Edema Macular/tratamiento farmacológico , Edema Macular/etiología , Microcirculación , Ranibizumab/uso terapéutico , Oclusión de la Vena Retiniana/complicaciones , Oclusión de la Vena Retiniana/diagnóstico , Oclusión de la Vena Retiniana/tratamiento farmacológico , Tomografía de Coherencia Óptica , Agudeza Visual
11.
PLoS One ; 15(10): e0240977, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33091078

RESUMEN

PURPOSE: To investigate factors associated with poor visual acuity (VA) in branch retinal artery occlusion (BRAO). METHODS: This was a retrospective cross-sectional study of 72 eyes with BRAO of 72 patients. For statistical comparison, we divided the patients into worse-VA (decimal VA < 0.5) and better-VA (decimal VA > = 0.5) groups. We examined the association of clinical findings, including blood biochemical test data and carotid artery ultrasound parameters, with poor VA. RESULTS: Median age, hematocrit, hemoglobin and high-density lipoprotein (HDL) differed significantly between the groups (P = 0.018, P < 0.01, P < 0.01, and P = 0.025). There was a tendency towards higher median IMT-Bmax in the worse-VA group (worse-VA vs. better-VA: 2.70 mm vs. 1.60 mm, P = 0.152). Spearman's rank correlation test revealed that logMAR VA was significantly correlated to IMT-Bmax (rs = 0.31, P < 0.01) and IMT-Cmax (rs = 0.24, P = 0.035). Furthermore, logMAR VA was significantly correlated to HDL level (rs = -0.33, P < 0.01). Multivariate logistic regression analysis revealed that IMT-Bmax (odds ratio [OR] = 2.70, P = 0.049), HDL level (OR = 0.91, P = 0.032), and female gender (OR = 15.63, P = 0.032) were independently associated with worse VA in BRAO. CONCLUSIONS: We found that increased IMT-Bmax, decreased HDL, and female sex were associated with poor VA in BRAO patients. Our findings might suggest novel risk factors for visual dysfunction in BRAO and may provide new insights into the pathomechanisms underlying BRAO.


Asunto(s)
Arterias Carótidas/patología , HDL-Colesterol/sangre , Oclusión de la Arteria Retiniana/sangre , Oclusión de la Arteria Retiniana/patología , Agudeza Visual/fisiología , Anciano , Grosor Intima-Media Carotídeo , Estudios Transversales , Ojo/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Trastornos de la Visión/sangre , Trastornos de la Visión/patología
12.
Bioconjug Chem ; 31(9): 2241-2251, 2020 09 16.
Artículo en Inglés | MEDLINE | ID: mdl-32840357

RESUMEN

Calpain activation induces retinal ganglion cell (RGC) death, while calpain inhibition suppresses RGC death, in animal studies. However, the role of calpain in human retinal disease is unclear. This study investigated a new strategy to study the role of calpain based on real-time imaging. We synthesized a novel fluorescent probe for calpain, acetyl-l-leucyl-l-methionine-hydroxymethyl rhodamine green (Ac-LM-HMRG) and used it for real-time imaging of calpain activation. The toxicity of Ac-LM-HMRG was evaluated with a lactate dehydrogenase cytotoxicity assay, retinal sections, and electroretinograms. Here, we performed real-time imaging of calpain activation in a rat model. First, we administered N-methyl-d-aspartate (NMDA) to induce retinal injury. Twenty minutes later, we administered an intravitreal injection of Ac-LM-HMRG. Real-time imaging was then completed with a noninvasive confocal scanning laser ophthalmoscope. The inhibitory effect of SNJ-1945 against calpain activation was also examined with the same real-time imaging method. Ac-LM-HMRG had no toxic effects. The number of Ac-LM-HMRG-positive cells in real-time imaging significantly increased after NMDA injury, and SNJ-1945 significantly lowered the number of Ac-LM-HMRG-positive cells. Real-time imaging with Ac-LM-HMRG was able to quickly quantify the NMDA-induced activation of calpain and the inhibitory effect of SNJ-1945. This technique, used as a companion diagnostic system, may aid research into the development of new neuroprotective therapies.


Asunto(s)
Calpaína/metabolismo , Carbamatos/farmacología , Activación Enzimática/efectos de los fármacos , Colorantes Fluorescentes/química , Retina/enzimología , Rodaminas/química , Animales , Calpaína/análisis , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Humanos , Fármacos Neuroprotectores/farmacología , Imagen Óptica , Ratas , Ratas Sprague-Dawley , Retina/efectos de los fármacos
13.
Acta Ophthalmol ; 98(6): e722-e729, 2020 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-32043815

RESUMEN

PURPOSE: To screen for anti-recoverin antibodies in elderly patients with retinitis pigmentosa (RP) with or without cancer and cross-sectionally characterize the seropositive patients clinically. METHODS: Serum from 75 RP patients who had been tested for mutations in a panel of 83 RP genes and 73 normal controls, all aged 50-80 years, were screened for anti-recoverin antibodies by Western blot using recombinant recoverin, retinal lysate from a marmoset and commercial anti-recoverin antibodies as a control. RESULTS: Three RP patients with typical pigmentary degeneration of the 75 (4.0%) were seropositive for anti-recoverin antibody. Pathogenic mutations were identified in two seropositive RP patients. All three patients had visual impairment since childhood and were diagnosed as RP by the age of 30. The severity of the retinopathy varied greatly among these three patients, ranging in visual acuity from light perception OU to 20/30 OU. Retinitis pigmentosa (RP) patients with a history of cancer were more likely to have anti-recoverin antibodies (3/14; 21.4%) than those without (0/61; 0%; p = 0.005, Fischer exact test). All 73 healthy controls with no history of cancer were also seronegative. CONCLUSION: Our results show that serum anti-recoverin antibodies can be detected in typical RP patients with identified pathogenic mutations and that a history of cancer may increase the risk of developing anti-recoverin antibodies.


Asunto(s)
Neoplasias/inmunología , Recoverina/antagonistas & inhibidores , Retinitis Pigmentosa/inmunología , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Estudios Transversales , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neoplasias/genética , Recoverina/sangre , Retinitis Pigmentosa/genética
14.
Sci Rep ; 9(1): 8875, 2019 06 20.
Artículo en Inglés | MEDLINE | ID: mdl-31221998

RESUMEN

This study evaluated age-related changes in the superficial and deep retinal capillary plexus (SCP and DCP, respectively) and in the foveal avascular zone (FAZ). SCP and DCP perfusion density (PD) were measured in optical coherence tomography angiography (OCTA) macular scans of 145 eyes of 145 healthy Japanese subjects, and findings were compared with SCP FAZ and clinical data. We found that age was negatively correlated with SCP and DCP PD (r = -0.17, P = 0.04 and r = -0.20, P = 0.02, respectively) and positively correlated with FAZ area (r = 0.18, P = 0.03). SCP and DCP PD were correlated with each other (r = 0.67, P < 0.001). FAZ area was negatively correlated with SCP PD, DCP PD and central macular thickness (CMT) (r = -0.18, P = 0.03; r = -0.25, P < 0.01; and r = -0.39, P < 0.001, respectively). FAZ was larger and CMT was lower (P = 0.01 and P < 0.001, respectively) in women than men. SCP and DCP PD were positively correlated with estimated glomerular filtration rate (r = 0.17, P = 0.03 and r = 0.24, P < 0.01, respectively). Multiple regression analysis confirmed that age independently affected DCP PD and FAZ (P = 0.02 and P < 0.01, respectively) and that CMT independently affected FAZ area (P < 0.001). Thus, normal subjects showed age-related decreases in macular PD and renal function. FAZ and CMT were related, suggesting that age-related changes in macular thickness also affect capillary vasculature.


Asunto(s)
Envejecimiento/fisiología , Capilares/diagnóstico por imagen , Fóvea Central/diagnóstico por imagen , Vasos Retinianos/diagnóstico por imagen , Agudeza Visual , Adulto , Anciano , Femenino , Voluntarios Sanos , Humanos , Japón , Masculino , Persona de Mediana Edad , Tomografía de Coherencia Óptica
15.
Org Lett ; 21(11): 4192-4196, 2019 Jun 07.
Artículo en Inglés | MEDLINE | ID: mdl-31120256

RESUMEN

Lithium binaphtholate, readily prepared from ( R)-3,3'-I2-BINOL and lithium tert-butoxide, efficaciously catalyzed the enantioselective aldol-Tishchenko tandem reaction of α-fluoroketones with aldehydes, achieving the enantioselective synthesis of 2-fluoro-1,3-diols with three contiguous stereogenic centers. Kinetic studies revealed that the aldol reaction and the subsequent hemiacetal formation are in equilibrium under the reaction conditions and that the lithium binaphtholate catalyst selectively promotes hydride shift of one of the eight stereoisomers to produce 2-fluoro-1,3-diols containing a tetrasubstituted fluorinated carbon center.

16.
Biochem Biophys Res Commun ; 506(3): 716-722, 2018 11 30.
Artículo en Inglés | MEDLINE | ID: mdl-30376992

RESUMEN

Bone remodeling is maintained through the balance between bone formation by osteoblasts and bone resorption by osteoclasts. Previous studies suggested that intracellular Ca2+ signaling plays an important role in the differentiation of osteoblasts; however, the molecular mechanism of Ca2+ signaling in the differentiation of osteoblasts remains unclear. To elucidate the effect of Ca2+ signaling in osteoblasts, we employed an optogenetic tool, blue light-activated Ca2+ channel switch (BACCS). BACCS was used to spatiotemporally control intracellular Ca2+ with blue light stimulation. MC3T3-E1 cells, which have been used as a model of differentiation from preosteoblast to osteoblast, were promoted to differentiate by BACCS expression and rhythmical blue light stimulation. The results indicated that intracellular Ca2+ change from the outside of the cells can regulate signaling for differentiation of MC3T3-E1 cells. Our findings provide evidence that Ca2+ could cause osteoblast differentiation.


Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Diferenciación Celular , Activación del Canal Iónico , Luz , Optogenética , Animales , Señalización del Calcio , Línea Celular , Espacio Intracelular/metabolismo , Activación del Canal Iónico/efectos de la radiación , Ratones Endogámicos C57BL , Osteoclastos/citología , Osteoclastos/metabolismo
17.
Front Neurosci ; 12: 561, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30174581

RESUMEN

As one of the ubiquitous second messengers, the intracellular Ca2+, has been revealed to be a pivotal regulator of various cellular functions. Two major sources are involved in the initiation of Ca2+-dependent signals: influx from the extracellular space and release from the intracellular Ca2+ stores such as the endoplasmic/sarcoplasmic reticulum (ER/SR). To manipulate the Ca2+ release from the stores under high spatiotemporal precision, we established a new method termed "organelle optogenetics." That is, one of the light-sensitive cation channels (channelrhodopsin-green receiver, ChRGR), which is Ca2+-permeable, was specifically targeted to the ER/SR. The expression specificity as well as the functional operation of the ER/SR-targeted ChRGR (ChRGRER) was evaluated using mouse skeletal myoblasts (C2C12): (1) the ChRGRER co-localized with the ER-marker KDEL; (2) no membrane current was generated by light under whole-cell clamp of cells expressing ChRGRER; (3) an increase of fluorometric Ca2+ was evoked by the optical stimulation (OS) in the cells expressing ChRGRER in a manner independent on the extracellular Ca2+ concentration ([Ca2+]o); (4) the ΔF/F0 was sensitive to the inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and (5) the store-operated Ca2+ entry (SOCE) was induced by the OS in the ChRGRER-expressing cells. Our organelle optogenetics effectively manipulated the ER/SR to release Ca2+ from intracellular stores. The use of organelle optogenetics would reveal the neuroscientific significance of intracellular Ca2+ dynamics under spatiotemporal precision.

18.
Methods Mol Biol ; 1668: 135-145, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28842907

RESUMEN

Optogenetic techniques are powerful tools for manipulating biological processes in identified cells using light under high temporal and spatial resolutions. Here, we describe an optogenetic training strategy to promote morphological maturation and functional development of skeletal muscle cells in vitro. Optical stimulation with a rhythmical frequency facilitates specific structural alignment of sarcomeric proteins. Optical stimulation also depolarizes the membrane potential, and induces contractile responses in synchrony with the given pattern of light pulses. These results suggest that optogenetic techniques can be employed to manipulate activity-dependent processes during myogenic development and control contraction of photosensitive skeletal muscle cells with high temporal and special precision.


Asunto(s)
Desarrollo de Músculos/fisiología , Fibras Musculares Esqueléticas/fisiología , Mioblastos/fisiología , Optogenética/métodos , Animales , Línea Celular , Luz , Potenciales de la Membrana , Ratones , Contracción Muscular , Imagen Óptica , Plásmidos , Cultivo Primario de Células , Sarcómeros/fisiología
19.
Nippon Ganka Gakkai Zasshi ; 121(2): 130-7, 2017 Feb.
Artículo en Japonés | MEDLINE | ID: mdl-30080003

RESUMEN

Purpose: To identify indices of visual improvement after conservative treatment for branch retinal vein occlusion (BRVO) with macular edema. Methods: We retrospectively reviewed the charts of 33 eyes of 33 patients with BRVO with macular edema. Inclusion criteria were 1) onset within 4 months, 2) decimal visual acuity of 0.05 to 0.5, and 3) minimum central subfield thickness (CST) of 250 µm. After 3 months of treatment with oral aspirin and kallidinogenase, the patients were divided into two groups: those with logarithmic minimum angle of resolution (logMAR) visual improvement of 0.3 or more (14 eyes) and less than 0.3 (19 eyes). We then compared systemic and ocular findings in the groups. Results: The groups differed significantly in logMAR improvement after 1 month (p<0.01) and in CST change after 1 month (p<0.05). Multiple logistic regression analysis showed that CST change after 1 month was a significant index of visual improvement (p<0.05). The cutoff value for visual improvement was -30 µm (sensitivity: 78.6, specificity: 68.4, positive predictive value: 64.7, negative predictive value: 81.3). Conclusion: A decrease in CST of more than 30 µm 1 month after conservative treatment indicates that visual acuity is likely to be improved after 3 months.


Asunto(s)
Edema Macular/fisiopatología , Edema Macular/terapia , Oclusión de la Vena Retiniana/fisiopatología , Oclusión de la Vena Retiniana/terapia , Anciano , Tratamiento Conservador , Femenino , Humanos , Edema Macular/complicaciones , Masculino , Persona de Mediana Edad , Oclusión de la Vena Retiniana/complicaciones , Estudios Retrospectivos , Resultado del Tratamiento , Agudeza Visual
20.
Nippon Ganka Gakkai Zasshi ; 119(6): 387-94, 2015 Jun.
Artículo en Japonés | MEDLINE | ID: mdl-26214889

RESUMEN

PURPOSE: To identify factors influencing visual outcomes in patients with hemorrhagic retinal arterial macroaneurysms (MA). METHODS: We retrospectively reviewed the charts of 13 eyes of 13 patients with hemorrhagic MAs. We evaluated factors. including age, blood pressure, ocular perfusion pressure, optic disc-MA distance, MA-fovea distance, the area of the hemorrhage, the time between onset and treatment, initial visual acuity, and the presence of subfoveal hemorrhage. Additionally, we measured the retinal cross-sectional area of the fovea with optical coherence tomography (OCT). RESULTS: There were significant differences in MA-fovea distance, area of the subretinal hemorrhage, and visual outcome in eyes with or without subfoveal hemorrhage (p < 0.05). Spearman's correlation analysis showed a significant negative correlation between visual outcome (logMAR) and disc-MA distance (rS = -0.61, p < 0.05), as well as MA-fovea distance (rS = -0.79, p < 0.01). A multivariate analysis showed an independent negative correlation between visual outcome and MA-fovea distance (Stdß = -0.66, t = 3.21, p < 0.01). In addition, there was a significant positive correlation between MA-fovea distance and the affected-/healthy-eye ratio of outer-retinal-layer cross-sectional area in the fovea (rS = 0.64, p < 0.05). The cutoff value of MA-fovea distance for subfoveal hemorrhage was 3000 microns, with a sensitivity of 100, specificity of 77.8, positive predictive value of 66.7 and a negative predictive value of 100. CONCLUSIONS: When hemorrhagic MAs are located closer to the fovea, the outer retinal layer is more severely affected and visual outcomes are poorer. Subfoveal hemorrhage should be considered even when it is not apparent, especially when the hemorrhagic MA is located within 3000 microns of the fovea.


Asunto(s)
Aneurisma/fisiopatología , Arteria Retiniana , Hemorragia Retiniana/etiología , Agudeza Visual , Anciano , Anciano de 80 o más Años , Aneurisma/patología , Humanos , Hemorragia Retiniana/patología , Estudios Retrospectivos
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA
...