Bacillus subtilis S1-4, isolated from chicken feather could efficiently degrade feathers by secreting several extracellular proteases. In order to get insight into the individual protease involved in keratin hydrolysis, a keratinase designed as BsKER71 was cloned and expressed in Bacillus subtilis WB600. In silico analysis revealed that BsKER71 protein contained a mature protein of 36.1 kDa. Further, purified BsKER71 could hydrolyze a variety of natural proteins, such as fibrous protein, collagen protein, casein, keratin and bovine serum albumin. In addition, this keratinase exhibited high enzyme activity in a wide range of pH and optimal pH of 10.0 and 9.0 in the hydrolysis of casein and keratin, respectively. Similarly, the optimal temperature was 55 °C and 50 °C for the hydrolysis of above two substrates, respectively. The hydrolytic activity was significantly inhibited by phenylmethanesulfonyl fluoride (PMSF), indicating the presence of serine residue in the active site. Moreover, ethylenediaminetetraacetic acid (EDTA) and phenanthroline moderately inhibited the hydrolytic activity. The catalytic activity was stimulated by Mg2+ and Ca2+, but greatly inhibited by Cu2+. Furthermore, several chemicals exhibited different effects on the hydrolysis of casein and keratin by BsKER71. These results provided a better understanding of BsKER71 from feather degrading bacterium B. subtilis S1-4. Keratin is the key structural component of outer coat of feather, hair, nail, horn, hoof and skin of animals. It is extremely stable and resistant to degradation due to its specific molecular structure Bradbury . For insBacillus, Streptomyces, Candida, Aspergillus and so on  and used as a host for recombinant expression of foreign gene. E. coli DH5α is used as a host for cloning.http://www.ncbi.nlm.nih.gov/BLAST) against the genome of B. subtilis S1-4 or GenBank (http://www.ncbi.nlm.nih.gov/). The isoelectric point (pI) and molecular weight prediction were determined by ProtParam (http://www.expasy.org/tools/protparam.html). Signal peptide was predicted at SignalP-4.1 Server . PROSITE was used to analyze the active sites. 26 published amino acid sequences of keratinases from various species (Bacillus licheniformis MKU 3 (gi: 67866986), Bacillus licheniformis PWD-1 (gi: 998767), Bacillus licheniformis BBE11-1 (gi: 407280558), Bacillus sp. MKR1 (gi: 336462511), Bacillus licheniformis MKU 2 (gi: 67866984), Bacillus licheniformis S90 (gi: 358680691), Bacillus licheniformis RG1 (gi: 46277126), Bacillus subtilis BF20 (gi: 164654845), Bacillus mojavensis (gi: 50363121), Bacillus licheniformis ER-15 (gi: 351000221), Bacillus licheniformis RPk (gi: 169883790), Bacillus licheniformis N5 (gi: 656355213), Bacillus licheniformis UTM107 (gi: 823327614), Bacillus licheniformis MZK05 (gi: 668730483), Bacillus licheniformis DS23 (gi: 300390464), Bacillus pumilus A1 (gi: 222353760), Bacillus pumilus KS12 (gi: 300429856), Bacillus tequilensis Q7 (gi: 846451730), Bacillus circulans DZ100 (gi: 511676716), Bacillus amyloliquefaciens K11 (gi: 893694191), Bacillus velezensis KJN-2 (gi: 443298521), Bacillus subtilis Egy-Ker (gi: 973694715), Bacillus subtilis Egy-KerM (gi: 973694712), Bacillus subtilis RSE163 (gi: 727929352), Bacillus subtilis B-3 (gi: 336109555), Bacillus subtilis YYW-1 (gi: 164664938)) were retrieved from GenBank database. Multiple sequence alignment was constructed using ClustalW program  and 3371-R1 (5′-ATTCGGTACCGGAACATCAGGATGCTGAC-3′). The PCR reaction mixture (50 μL) contained 400 nmol/L of each primer, 100 μmol/L dNTP, 50 ng gDNA of S1-4, and Pfu DNA polymerase. The amplification conditions were as follows: initial denaturation at 95 °C for 2 min, 30 cycles at 94 °C for 30 s, 52 °C for 30 s, 68 °C for 2 min, and a final extension at 72 °C for 10 min. The PCR products were then purified using SanPrep DNA gel extraction kit  following the manufacturer’s instructions, and digested by PstI and KpnI restriction enzymes . The resultant PCR products were cloned into E. coli–B. subtilis shuttling vector pSUGV4 digested with same restriction enzymes by DNA ligation and transformation. The resulting recombinant plasmid pSUGV4-KER71 was confirmed by PCR amplification and DNA sequencing.Based on the genome of B. subtilis WB600 as previously described , fibrin , collagen , BSA , or casein (10 mg/mL)  was mixed with 5 μL (4.8 μg) of purified BsKER71 in 20 μL boric acid–NaOH buffer (pH 9.6). After incubation for 20 min at 50 °C, proteolytic reaction was terminated by heating at 95 °C for 10 min. Then, samples were directly loaded onto 12% SDS-PAGE.The keratinase activity was determined as previously described using keratin powder  as substrate  on the catalytic activity was examined as described above using casein and keratin as substrate. To determine thermal stability, enzyme sample was diluted in boric acid–NaOH buffer (pH 9.6), and incubated at selected temperatures for 20 min. Each enzyme sample was immediately transferred on ice. Residual activity was determined as described above using casein and keratin as substrate.2+, Co2+, Mg2+, Ca2+, Mn2+, Ni2+, Cu2+), and chemical reagents  on the hydrolytic activity of BsKER71, each compound at the indicated concentration was added to assaying mixture. The hydrolytic reaction was performed following the standard procedure as described above. All assays were performed in triplicates.To determine the effect of protease inhibitors , metal ions (ZnB. licheniformis and gi: 336109555 from B. subtilis) were aligned with the genomic data of the B. subtilis S1-4 by local BLASTP. A homologous sequence was identified in the genome of S1-4. The entire coding sequence (designed as Bsker71) was 1146 bp in length (accession number: MN256128), encoding a deduced protein of 381 amino acids with a molecular weight of 39.45 kDa and theoretical isoelectric point of 9.04  and B. subtilis YYW-1 (gi: 164664938), but appeared to be distinct from keratinase (e.g. gi: 998767) of B. licheniformis PWD-1  . When the temperature was above 55 °C, hydrolytic activity was rapidly decreased Fig. c.It was observed that PMSF almost completely inhibited the hydrolytic activity, indicating that BsKER71 is a serine protease Fig. . However2+ (2 mmol/L) and Ca2+ (2 mmol/L) promoted the hydrolytic activity of BsKER71 to some extent when casein and keratin were used as substrates. In contrast, Cu2+ (2 mmol/L) almost completely inhibited the enzymatic activity. The other metal ions exhibited only slight inhibition, maintaining > 90% activity. However, no obvious difference was observed for both substrates.The effect of metal ions on the catalysis of BsKER71 was shown in Table 2SO3. With the increased concentration of the surfactants to 5 mmol/L, slight inhibition was observed, especially 5 mmol/L of DMSO exhibited significant inhibition      (Muhsin and Aubaid Aspergillus parasiticus (36 kDa)  or increased (1 mmol/L) by DTT. In addition, it was observed that DTT might have almost no influence on the enzyme activity of BsKER71. This was similar to that of keratinase KERAB from Streptomyces sp. strain AB1 (Jaouadi et al. P. marquandii and D. microspores (Gradisar et al. It has been reported that the addition of DTT resulted in increased keratinase activity through the breakage of disulfide bonds in tightly packed keratins (Riffel et al. B. subtilis S1-4 was cloned, expressed and characterized in this study. The purified enzyme could hydrolyze several natural proteins and exhibited temperature adaptability and higher enzyme activity at alkaline pH. Results indicated that BsKER71 had a certain potential in safe, economical and environment-friendly utilization of feather wastes.In conclusion, a novel keratinase BsKER71 from Additional file 1: Figure S1. A typical elution profile of the recombinant keratinase by gel filtration chromatography column. Peak 1: unknown protein; Peak 2: BsKER71 protein; Red peak: ammonium sulfate.Additional file 2: Figure S2. The protease activity test of eluting samples from peak 2 on the milk containing agarose plate.Additional file 3: Figure S3. Quantitative determination of BsKER71 protein on SDS-PAGE. Lane 1, 2, 3, 4, 5 correspond to 0.5, 1.0, 2.0, 3.0, and 4.0 μg BSA, respectively; lane 6 and 7 were loaded with 2.5 μL and 5.0 μL purified BsKER71 protein.


Cataract is an eye disease with clouding of the eye lens leading to disrupted vision, which often develops slowly and causes blurriness of the eyesight. Although the restoration of the vision in people with cataract is conducted through surgery, the costs and risks remain an issue. Botanical drugs have been evaluated for their potential efficacies in reducing cataract formation decades ago and major active phytoconstituents were isolated from the plant extracts. The aim of this review is to find effective phytoconstituents in cataract treatments in vitro, ex vivo, and in vivo. A literature search was synthesized from the databases of Pubmed, Science Direct, Google Scholar, Web of Science, and Scopus using different combinations of keywords. Selection of all manuscripts were based on inclusion and exclusion criteria together with analysis of publication year, plant species, isolated phytoconstituents, and evaluated cataract activities. Scientists have focused their attention not only for anti-cataract activity in vitro, but also in ex vivo and in vivo from the review of active phytoconstituents in medicinal plants. In our present review, we identified 58 active phytoconstituents with strong anti-cataract effects at in vitro and ex vivo with lack of in vivo studies. Considering the benefits of anti-cataract activities require critical evaluation, more in vivo and clinical trials need to be conducted to increase our understanding on the possible mechanisms of action and the therapeutic effects. The ocular lens is located at the anterior segment of the eye that, together with the cornea, provides the refractive power of the eye. The mature lens is composed of a core of primary lens fiber cells, layers of secondary lens fiber cells, and one layer of anterior lens epithelial cells, which covers the anterior surface of the lens . The majCataracts are the most common cause of vision loss in people over the age of 40 and are the leading cause of blindness in the world . CataracThere have been many compounds evaluated for their potential efficacies in reducing cataract formation. In this article, we focus on active ingredients derived from plants. Phytoconstituents are a trove of often structurally complicated compounds with interesting biological functions. They themselves or their derivatives have always been important sources of pharmacologically active agents. To provide a comprehensive review of potentially useful anti-cataract phytoconstituents, we searched, selected, and extracted the appropriate information from published literature according to the following procedures. We feel that, by listing the comprehensive collection of phytocontituents in one place, this manuscript serves as an overview and perhaps an inspiration to prompt additional studies in this important research area. Collaborative efforts between phytochemists and cataract researchers are promisingly fruitful.2O2-induced opacity/cataract, congenital/juvenile cataract, transgenic/knockout mice with cataract, diabetic cataract, spontaneous cataract, isolated phytoconstituents, medicinal plants.Literature search of articles published from January 2008 to December 2017 was performed. We searched the databases of Pubmed, Science direct, Google Scholar, Web of Science and Scopus using different combinations of keywords: lens epithelial cells, sodium selenite-, ultraviolet radiation-, steroid induced, oxygen-, HThe selection of the manuscripts was based on the following inclusion criteria: isolation of phytoconstituents from plants, and selection of phytoconstituent(s) with the most potent anti-cataractogenesis activities. Exclusion of manuscripts from this review involves synthesized/commercialized compounds, phytoconstituents screening without isolated compounds, activities with extracts only and isolated phytoconstituents without names or articles that did not meet the inclusion criteria. The selection process is summarized in 50, treatment duration (in vivo) and isolated phytoconstituent(s) with the strongest activity(s), as well as their structural formula. The extracted data are presented in All the selected manuscripts were analyzed for year of publication, plant species, family, part of plant, solvent extraction, isolation method, isolated phytoconstituents, anti-cataract activities , route of administration (in vivo), dose or concentration for IC2O2)-, xylose-, galactose-induced lens opacity, aldose reductase (AR) activity assay, and advanced glycation end products (AGE) formation. In vivo models include sodium selenite-, ultraviolet (UV) radiation-, and steroid-induced cataracts. These models have been widely used to study the mechanisms of cataract and serve as the screening platform of anti-cataract therapies with the long-term goal to treat cataract in humans.There are a large number of in vitro and in vivo models that mimic certain aspects of the pathophysiological features of human cataracts. They have been used to demonstrate the potential therapeutic effects of phytochemicals. In this section, we describe the most commonly used models in order to aid the understanding and appraisal of results. During our literature search, most of the phytochemicals were tested in in vitro or ex vivo models only. Only a dozen or so were assessed in in vivo cataract models. Nevertheless, for completion’s sake, we list both in vitro and in vivo models. In vitro models discussed are hydrogen peroxide (H2O2) is the major reactive oxygen species (ROS). H2O2 is mainly generated in vivo by the detoxification of superoxide (O2−) radical by superoxide dismutase (SOD) through the dismutation reaction -6″-O-acetyl-β-d-glucopyranoside was isolated from Sideritis brevibracteata (Dağ çayı) [50 value of 0.66 µM [Hypolaetin 7-ağ çayı)  and appeağ çayı) ,95. This 0.66 µM .29H36O16 (640.58 g/mol).Molecular formula: C2O  seeds extract of Paulownia coreana  at room temperature for 72 h. Paulownia coreana is long cultivated in Eastern Asia, particularly Korea and has been used traditionally in medicines for certain ailments [50 value of 9.72 µM [Isocampneoside II is an active phenylethanoid glycoside isolated from acetone-Hailments . A total 9.72 µM .22H22O12 (478.406 g/mol), Melting point: 168–172 °C.Molecular formula: CCochlospermum religiosum  has been reported to possess anticataract activity [C. religiosum yielded isorhamnetin-3-glucoside. This bioactive compound was obtained as yellow needles and identified as flavonoids with yellowish orange color in alkali, pink in Mg-HCl and reaction with Fe3+ gives olive green color. Isorhamnetin-3-glucoside at the concentration of 25 µg/mL (52 µM) inhibited further formation of vacuoles and opacity on sodium selenite-induced lens opacity of rat pups. The antioxidant property of isorhamnetin-3-glucoside was suggested to complement its anticataract activity [activity . Purificactivity .15H10O6 (286.23 g/mol), Melting point: 276–278 °C.Molecular formula: CLitsea japonica (Thunb.) Juss. (hamabiwa) showed the most potent against RLAR inhibitory activity with IC50 value of 1.10 µM among all phytoconstituents isolated [Agrimonia pilosa Ledeb , Allium victorialis (victory onion), and Paulownia coreana with IC50 values of 15.2 µM (RLAR) [50 of 36.01 µM from Allium victorialis [Kaempferol isolated from isolated . The samM (RLAR) , 1.10 µMM (RLAR) , and 45.M (RLAR) , respecttorialis .28H32O15 (608.549 g/mol), Melting point: 251–253 °C.Molecular formula: CViola hondoensis W. Becker et H Boss (ri ben qiu guo jin cai) for its AR inhibitory activity. This plant is widely distributed in southern Korea and has been used to as traditional medicine in the form of expectorant. AR activity-guided isolation using column chromatography on a silica gel and gel filtration column afforded kakkalide. Kakkalide significant inhibited AR from Sprague-Dawley rat lenses at an IC50 of 0.34 µg/mL (0.56 µM), more potent than that of the positive control, tetramethylene glutaric acid (IC50 = 0.48 µg/mL) [Kakkalide was isolated from 8 µg/mL) .30H48O4 (472.69 g/mol).Molecular formula: CGanoderma lucidum  from a thorough fractionation process [50 of 19.1 µM compared to all other reported isolated secondary metabolites including ganoderic acid Df (IC50 = 22.8 µM) [50 = 43.8 µM) [50 = 110.1 µM) [Lucidumol A is a new triterpenoid isolated from the ethanolic extract of the fruiting body of  process . Obtaine process , lucidum22.8 µM) , ganoder43.8 µM) , ganoder10.1 µM) , and oth30H50O (426.72 g/mol), Melting point: 120–122 °C.Molecular formula: CMusa sp. var. Nanjangud rasa bale (banana) [Vernonia cinereal (purple fleabane) [50 of 1.53 µg/mL (3.6 µM) [Lupeol, a pentacyclic triterpenoid has been isolated from the ethanolic flower extract of (banana)  and methleabane)  with ant(3.6 µM) . Similar(3.6 µM) . The pot(3.6 µM) .15H10O6 (286.24 g/mol), Melting point: >320 °C.Molecular formula: CPlatycodon grandiflorum  [Artemisia montana [Perilla frutescens (L.)  [Sinocrassula indica (Chinese crassula) [Vitex negundo) demonstrated 80% transparency of the lenses with minor cortical vacuolization and opacity suggesting that the anticataractogenic effect was supported by the antioxidant property based on significant decrease in various antioxidant activities tested [Platycodon grandiflorum was identified as the highest inhibition with an IC50 of 0.087 µM (RLAR) [50 value increases slightly higher to 0.45 µM (Sinocrassula indica) in the same activity [Perilla frutescens (L.) of different parts showed different values in RLAR  [50 6.34 µM) [Artemisia montana) was found to suppress RLAR activity at an IC50 0.19 µM [Platycodon grandiflorum (Jacq.) exhibited comparable potent inhibitory effect of AGE (IC50 = 16.6 µM) with chlorogenic acid methyl ester (IC50 = 12.9 µM) [Chinese bellflower) , Vitex ne vitex) , Artemis montana , Perillaperilla) ,113,114 rassula) . The sels tested . In compM (RLAR) . The IC5activity . However6.34 µM) , almost  0.19 µM . Luteoli12.9 µM) .21H20O11 (488.38 g/mol), Melting point: >195 °C.Molecular formula: CO-β-d-glucopyranoside was isolated from the leaves extract of Stauntonia hexaphylla (Thunb.) Decne. (stauntonia vine), traditionally used as folk medicine in China, Japan and Korea. Luteolin-7-O-β-d-glucopyranoside showed the highest potent inhibitory activity of RLAR at IC50 value of 7.34 µM among other isolated secondary metabolites. Inhibition was reported 2.4 times higher compared to that of quercetin. This data was substantiated by inhibition of AGE at IC50 value of 117.8 µM and it was suggested that the presence of sugar position in flavonoids enhances the activity [Luteolin-7-20H24NO4+ (342.41 g/mol).Molecular formula: CTinospora cordifolia  [Coptidis rhizome  [50 value at 3.6 µM from isolation of Tinospora cordifolia. Further analysis showed that magnoflorine inhibited 72.3% of galactose-induced polyol accumulation [Coptidis rhizome possessed marginal inhibition against RLAR with 18% inhibition at a concentration of 146 µM. At this point, it is not clear if the very significant differences in efficacies and potencies were due to technical differences in isolation and/or biological assay.Magnoflorine was isolated from , giloy)  and Coptng lian)  for inhimulation . Neverth26H26O12 (530.50 g/mol).Molecular formula: CO-caffeoylquinate or also known as 3,5-di-O-caffeoylquinic acid methyl ester was isolated from the flowers of Erigeron annuus [Xanthium strumarium [Erigeron annuus methanolic flower extract afforded methyl-3,5-di-O-caffeoylquinate with yellow gum appearance at percentage yield of 0.0075% [O-caffeoylquinate from both plants suppressed RLAR activity at IC50 values of 0.3 to 0.81 µM. Significant results (most potent) were also observed with further assays in RHAR and galactitol accumulation in rat lenses ex vivo from Xanthium strumarium at IC50 value of 0.67 µM and 117 µg/lens wet weight, respectively [Methyl-3,5-di-n annuus  and fruirumarium  with hig 0.0075% . Isolateectively .24H24O10 (472.44 g/mol).Molecular formula: CPrunus mume (Japanese apricot), mumeic acid-A was found to be the most potent inhibitor from all isolated secondary metabolites. The IC50 concentration of mumeic acid-A (IC50 = 0.4 µM) was almost twice that of chlorogenic acid (IC50 = 0.7 µM) as the positive control [In an attempt to obtain inhibitors of RLAR from  control ,120.16H12O5 (360.32 g/mol), Melting point: 294–296 °C.Molecular formula: CPueraria lobata has for long been used in Far Eastern Asia countries as traditional medicine [50 value of 22.2 µM, much lower than the positive control, 3,3-Tetramethyleneglutaric acid (IC50 = 28.8 µM). This data was substantiated with prevention of xylose-induced lens opacity in a dose-dependent manner, with the highest dose at 15 µM [The roots of medicine . The isoat 15 µM .21H20O12 (464.38 g/mol).Molecular formula: CO-β-d-glucoside was isolated from Petasites japonicus  and Stauntonia hexaphylla as inhibitor for RLAR and AGE activities. It inhibited RLAR activity at IC50 values between 2.21 and 10.4 µM [50 = 1 mM) [Quercetin-3- 10.4 µM ,122. In  = 1 mM) .21H20O11 (448.38 g/mol), Melting point: 177–183 °CMolecular formula: CSmilax china L. (China root), Agrimonia pilosa Ledeb, Allium victorialis var. platyphyllum, and Melastoma sanguineum using various isolation methods. It was tested for RLAR and AGE inhibitory assays and found that quercitrin possesses significant inhibitory actions for both activities among all isolated phytoconstituents in these plants. The IC50 values were reported at 0.17 to 0.56 µM (RLAR) and 42.0 to 58.0 µM (AGE) [Melastoma sanguineum showed IC50 value of 0.16 µM (RLAR) and 25.11 µM (AGE), respectively [Quercitrin, a glycosylation of quercetin at C-3, was isolated from few botanicals including µM (AGE) ,100,123.ectively .19H17N3O2 (319.36 g/mol), Melting point: 196 °C.Molecular formula: CEvodia rutaecarpa Bentham (Rutaceae) (wu zhu yu) is part of the Kampo-herbal medicine in Japan and has been used to relive digestion as well as painkiller. Various compounds have been isolated from this plant especially rhetsinine showed potent inhibitory activity against RLAR at an IC50 value of 24.1 µM. Rhetsinine was also reported to inhibit sorbitol accumulation in human erythrocyte by almost 79.3% at 100 µM [t 100 µM .18H16O8 (360.32 g/mol), Melting point: 171–175 °C.Molecular formula: CSalvia grandifolia (da ye shu wei cao) recorded the lowest IC50 (0.30 µM) in RLAR activity [50 values of rosmarinic acid isolated from other plants were 2.77 µM  [Colocasia esculenta (L.) Schott; taro) [Perilla frutescens L.) [Perilla frutescens L.) [Prunella vulgaris L.) with potent activity on galactitol accumulation in rat lenses, but low inhibitor of AGE (20.7%) for Prunella vulgaris L. [Rosmarinic acid, a conjugation of caffeic acid and 3,4-dihydroxyphenyllactic acid is mainly isolated from the family of Lamiaceae with good anti-cataract activity. Isolated rosmarinic acid from activity  comparedlf-heal) , 5.38 µMt; taro) , and 11.cens L.) , respectcens L.)  and 18.6garis L. .10H8O4 (192.16 g/mol), Melting point: 204–205 °C.Molecular formula: CMagnolia fargesii (Shin-i) air-dried buds yielded five compounds with scopoletin showing the most potent in RLAR, AGE and xylose-induced lens opacity assays [50 value of 2.9 µM, approximately 327 times more potent compared to the positive control (IC50 = 961 µM). Similarly, in the RLAR activity, scopoletin showed marked inhibitory activity with an IC50 value of 22.5 µM. This was substantiated by the suppression of lens opacity to 72.9% (25 µM) after three days of xylose treatment [50 concentration of RLAR activity was observed for scopoletin isolated from Angelica gigas  with an IC50 value of 2.6 µM [Artemisia montana showed higher IC50 value at 64.5 µM for the same activity [The bioassay-guided fractionation of methanol extract of y assays . Scopolereatment . A lowerf 2.6 µM  showing activity ,129.20H16O6 (352.34 g/mol), Melting point: 131–134 °C.Molecular formula: CGlycyrrhiza sp.) [Glycyrrhiza uralensis with semilicoisoflavone B showed the most potent inhibition of RLAR and RHAR activities. Both inhibition rates were recorded at IC50 values of 1.8 and 10.6 µM, respectively. Unlike γ,γ-dimethylallyl type prenylated isoflavonoids, semilicoisoflavone B containing γ,γ-dimethylchromene ring on the aromatic ring inhibited AR more strongly. In kinetic analysis of AR inhibition, semilicoisoflavone B did not bind to any substrate and NADPH binding regions of RHAR. Ex vivo analysis showed that this compound highly inhibited sorbitol accumulation in rat lenses incubated with high glucose by 47.0% [Semilicoisoflavone B is mostly found in roots and rhizomes of licorice species (iza sp.) . In searby 47.0% .15H10O5 (270.24 g/mol), Melting point: 295–303 °C.Molecular formula of sulfuretin: C15H12O5 (272.25 g/mol), Melting point: 216 °C.Molecular formula of butein: CRhus verniciflua (lacquer tree) produced nine secondary metabolites with sulferetin and butein as the most potent phytoconstituents for AGE and RHAR, respectively. Sulferetin was isolated as white to off-white crystalline powder [50 value of 124 µM, 11 times lower than aminoguanidine (IC50 = 1450 µM). The RHAR inhibitory activity of butein was reported at IC50 = 0.7 µM [The AR and AGE guided isolation of ethanolic bark extract of e powder  and inhi= 0.7 µM . The eff= 0.7 µM  and hydr= 0.7 µM .9H10O5 (198.17 g/mol), Melting point: 206–208 °C.Molecular formula: CO-methylated trihydroxybenzoic acid monomer extracted from Herba dendrobii (shi hu). Herba dendrobii, found in the stem of many orchid species of the Dendrobium genus, has been used to improve vision centuries ago [Herba dendrobii improves survival of high-concentration d-galactose-injured human LEC with inhibition ratio of 20.3%. Rat lens turned clear to Grade 0 after 90 days of treatment. Syringic acid inhibits AR activity in a dose-dependent manner with an IC50 value of 213.17 µg/mL (1075.7 µM). Data suggest that syringic acid downregulates the expression of mRNA of AR [Magnolia officinalis was weaker with less than 10% of inhibition [Syringic acid is a phenolic compound and a naturally occurring ries ago . SyringiNA of AR . Howeverhibition .22H22O10 (446.40 g/mol), Melting point: 243 °C.Molecular formula: CEnicostemma hyssopifolium  methanol extract after repeated column chromatography over silica gel. This compound reacts with ferric chloride and turned greenish brown color as a confirmation test for flavonoids. RLAR activity was significantly inhibited by swertisin at an IC50 value of 0.71 µg/mL  indicating a higher inhibition compared to the other compound isolated, swertiamarin (IC50 = 7.59 µg/mL). This compound was also found to suppress polyol accumulation (41.7%) in lenses cultured in a galactitol medium [Swertisin appears as pale yellow powdery crystals and isolated from l medium .21H10O13 (470.29 g/mol), Melting point: 177–183°C.Molecular formula: CSyzygium cumini (L.) Skeels lead to the isolation of six phytoconstituents with valoneic acid dilactone showed the highest activity against RLAR inhibitory activity at IC50 value of 0.075 µM [The repeated column chromatography and preparative HPLC of seed methanolic extract of 0.075 µM . ValoneiDespite the success in surgical replacement of the cataractous lens with an artificial intraocular lens, discovery of pharmacological prevention and treatment of this blinding disorder has been an earnest, continuous effort in ophthalmology research. In this review manuscript, we summarize findings of phytoconstitutents and their pharmacological effects as potential anti-cataract agents. The large number of interesting compounds is exciting. It raises hope that clinically useful medication may have a good chance to be derived from this sizable collection of chemicals with diverse structural scaffolds.O-galloyl-β-d-glucose inhibits AGE formation with an IC50 of 2 M. Both 1,3,5,8-tetrahydroxyxanthone and 2′′,4′′-O-diacetylquercitrin inhibit AR with IC50 values below 0.1 M. However, a major limitation of the listed compounds is that although they have been shown to have the appropriate biological actions in a variety of in vitro or ex vivo assays, many of them were not tested in animal cataract models. Additionally, a few have been evaluated in only one animal model. Without relevant in vivo data, it is obviously very difficult to develop the compounds into meaningful treatments for cataract patients.Many of the compounds have potent and efficacious in vitro pharmacological activities that are consistent with potential anti-cataract effects. For example, 1,2,3,6-tetra-In addition to the lack of in vivo data, there are other challenges facing development of anti-cataract pharmaceuticals. For example, cataract medication has to compete with the very successfully and generally affordable (as least in developed countries) surgical procedure. Moreover, pharmacological prevention of cataract formation is expected to require a long-term, likely multi-year, administration of medicine, which, to some, is undesirable. Overcoming these challenges necessitates careful considerations of drug safety, convenience of administration, and cost. These concerns may have previously prohibited the development of certain agents. Nonetheless, we feel that phytoconstituents are advantageous compared to conventional synthetic drugs. Many societies have been using plant products from where some of the ingredients are derived for centuries, indicating long-term safety and acceptance. The development path and clinical use will be similar to vitamins and phytochemicals such as lutein and zeaxanthine. If proven safe, cost-effective, and most importantly, efficacious in preventing or reversing cataract formation, phytoconstituents can be a revolutionary approach in the treatment of cataract.Despite the success in surgical replacement of the cataractous lens with artificial intraocular lens, pharmacological prevention and treatment of this blinding disorder have been an earnest, continuous effort in ophthalmology research. In this review manuscript, we summarize findings of 56 entries of phytoconstitutents and their pharmacological effects as potential anti-cataract agents. The large number of interesting compounds is exciting. It raises hope that clinically useful medication may have a good chance to be derived from this sizable collection of chemicals with diverse structural scaffolds.O-galloyl-β-d-glucose inhibits AGE formation with an IC50 of 2 µM. Additionally, both 1,3,5,8-tetrahydroxyxanthone and 2′′,4′′-O-diacetylquercitrin inhibit AR with IC50 values below 0.1 µM. However, a major limitation of the listed compounds is that although they have been shown to have the appropriate biological actions in a variety of in vitro or ex vivo assays, many of them were not tested in animal cataract models. And a few have been evaluated in only one animal model. Without relevant in vivo data, it is obviously very difficult to develop the compounds into meaningful treatments for cataract patients. We feel that, by listing the comprehensive collection of phytocontituents in one place, this manuscript serves as an overview and perhaps an inspiration to prompt additional studies in this important research area. Collaborative efforts between phytochemists and cataract researchers are promisingly fruitful.Many of the compounds have potent and efficacious in vitro pharmacological activities that are consistent with potential anti-cataract effects. For example, 1,2,3,6-tetra-In addition to the lack of in vivo data, there are other challenges facing development of anti-cataract pharmaceuticals. For example, cataract medication has to compete with the very successfully and generally affordable (as least in developed countries) surgical procedure. Moreover, pharmacological prevention of cataract formation is expected to require a long-term, likely multi-year, administration of medicine, which, to some, is undesirable. Overcoming these challenges necessitates careful considerations of drug safety, convenience of administration, and cost. These concerns may have previously prohibited the development of certain agents. Nonetheless, we feel that phytoconstituents are advantageous compared to conventional synthetic drugs. Many societies have been using plant products where some of the ingredients are derived from for centuries, indicating long-term safety and acceptance. The development path and clinical use will be similar to vitamins and phytochemicals such as lutein and zeaxanthine. If proven safe, cost-effective, and most importantly, efficacious in preventing or reversing cataract formation, phytoconstituents can be a revolutionary approach in the treatment of cataract.


The average optical transmittance of the films prepared at F/Sn of 0–0.5 was over 90%, and it decreased to 84.4% for the film prepared at F/Sn = 1. The hardness (H) and Young’s modulus (E) of the FTO films increased when the F/Sn ratios increased from 0 to 0.5, reaching maximum values of H = 12.3 ± 0.4 GPa, E = 131.7 ± 8.0 GPa at F/Sn = 0.5. Meanwhile, the H and E reduced considerably when the F/Sn ratio further increased to 1.0, following the inverse Hall-Petch effect approximately, suggesting that the grain boundary effect played a primary role in manipulating the nanomechanical properties of the FTO films. Furthermore, favorable mechanical properties with large fH/E and Fluorine-doped tin oxide (FTO) thin films were deposited on glass substrates using ultrasonic spray pyrolysis (USP) at a fixed substrate temperature of 400 °C and various Fluorine/Tin (F/Sn) atomic ratios of 0, 0.1, 0.5, and 1.0. Effects of F/Sn atomic ratios on structural-morphological, compositional, electrical, optical, and nanomechanical properties of the FTO thin films were systematically studied. The FTO films exhibited a tetragonal structure with preferred orientations of (110), (200), and (211), and polycrystalline morphology with spear-like or coconut shell-like particles on the surfaces. The presence of F-doping was confirmed by XPS results with clear F1s peaks, and F-concentration was determined to be 0.7% for F/Sn = 0.1 and 5.1% for F/Sn = 0.5. Moreover, the resistivity of FTO films reduced remarkably from 4.1 mΩcm at F/Sn = 0 to 0.7 mΩcm at F/Sn = 1, primarily due to the corresponding increase of carrier concentration from 2 × 10 FFTO thin films have been extensively prepared by various methods, such as atmosphere pressure chemical vapor deposition (APCVD) ,15,16, sBesides the structural-morphological, electrical, and optical properties, the nanomechanical properties of TCO films are important parameters that determine the performance of devices involving TCO films. It is essential for designing stress-free thin-film, semi-transparent multi-layer structures and top-emitting organic light-emitting displays that involve one or more layers of TCO films on both rigid and flexible substrates . The YouIn this study, we successfully employed ultrasonic spray pyrolysis  to fabricate FTO thin films on glass substrates. Especially, the atomizer was an ultrasonic nebulizer, which was originally used for treatment of respiratory disease (particularly asthma) using high-frequency vibrations to turn liquid medication into a mist. The structural, morphological, compositional, electrical, optical, and nanomechanical properties of polycrystalline FTO thin films prepared at F/Sn atomic ratios of 0, 0.1, 0.5, and 1.0 were systematically studied. The results in this study will provide a strategy for fabricating high-quality FTO thin films with the enhanced both desired material and nanomechanical properties for applications.2 thin films were deposited on Corning glass substrates (15 × 15 × 1 mm3) at a substrate temperature of 400 °C and a mixed oxygen-argon carrier gas at a flow rate of 1.5 L/min, and at various F/Sn atomic ratios of 0, 0.1, 0.5, and 1.0 using USP. In a typical procedure, 5.64 g SnCl2·2H2O was dissolved in 25 mL distilled water. To improve the solubility of the solution, 2 mL of HCl was added to the solution and stirred vigorously (~30 min) until the solution turned transparent. Then, NH4F with appropriate weights were added to the solution to introduce different F-doping levels at F/Sn = 0, 0.25, 0.5, and 1. The solution was then stirred at 600 rpm for approximately 30 min until the solution turned transparent. The product solution was placed in an ultrasonic nebulizer reactor, which can produce an aerosol with a controlled droplet size around 0.5–5 μm depending on the precursor solution. The experimental apparatus consists of a specific homemade ultrasonic atomizer, spray gun, and graphite hotplate (see Florine-doped SnOlate see b inset. late see b inset. α radiation (λ = 1.5406 Å) in 2θ-θ configuration. Surface morphology and film thickness were examined using field-emission scanning electron microscopy  through plane-view and cross-sectional images, respectively. The surface feature and roughness of FTO thin films were characterized through atomic force microscopy . The center line average (aR) was used to present surface roughness. Detailed structural information at atomic scale of a selected FTO film was obtained from high-resolution scanning transmission electron microscope (HRTEM) images  operated at 200 kV. The surface chemical composition and F-doping concentration of FTO thin films were characterized using X-ray photoelectron spectroscopy  with the X-ray source . The binding energies obtained in the XPS analysis were standardized using C1s at 285.0 eV. XPS curve fitting was performed using the freeware XPSPEAK 4.1 with the Shirley background subtraction, and assuming a Gaussian-Lorentzian peak shape. In-plane electrical conductivity, carrier concentration (n) and mobility (μ) were measured at room temperature by using a Hall system  with van der Pauw geometry.The crystal structure of FTO thin films was determined through X-ray diffraction  using CuK® system, MTS Cooperation, Nano Instruments Innovation Center, Oak Ridge, TN, USA). Nanoindentation measurements were made employing a triangular pyramid Berkovich diamond indenter with curvature radius of 50 nm. The continuous stiffness measurement technique was used in the nanoindentation tests [−1 until an indent depth of 80–88 nm was achieved and held for 5 s at the peak load. Finally, the indenter was withdrawn with the same strain rate until 10% of the peak load was reached. At least 20 indents were performed on each sample. Each indentation was separated by 50 μm to avoid possible interferences between neighboring indents. The analytical method proposed by Oliver and Pharr was used to determine the hardness and Young’s modulus of the FTO thin films [Nanomechanical properties  of the FTO thin films were obtained by nanoindentation tests , (200), and (211). The grain sizes (D) of the films were estimated by using the Scherrer equation D = 0.9λ/βcosθ, where λ, β, and θ are the X-ray wavelength, full width at half maximum of the FTO (110)- oriented peak, and Bragg diffraction angle, respectively. The estimated D values of FTO thin films increased from 29.6 nm at F/Sn = 0 to 42.7 nm at F/Sn = 0.5, and then decreased to 35.1 nm at F/Sn = 1, as shown in 2 film to 27.1 nm for the 12 wt.% F-doped SnO2 film. Noticeably, there is a small peak at ~31.7° for the film prepared at F/Sn = 0.5 that suggests the presence of Sn3O4 impurity phase in the film [3O4 impurity phase is also detected in the FTO films prepared by USP [d by USP . D result estimated by XRD, the particle sizes observed from SEM images appeared to be increased for F/Sn in 0–0.5 range and decreased for F/Sn = 1.0. Noticeably, some large clusters composed of smaller particles were found on the surface of the highest doping level film . It is worthy of note that a particle size (observed from SEM images) can be larger than the grain  size (obtained from XRD result), as it may consist of number of grains and even include amorphous regions. As shown in the insets of aR) of 11.9–20.7 nm , carrier mobility (μ), and carrier concentration (n)) of the FTO thin films grown at various F/Sn atomic ratios are shown in n increased substantially from 2 × 1020 to 13 × 1020 cm−3 as F/Sn increased from 0 to 0.5, and then it decreased slightly to 11.6 × 1020 cm−3 at F/Sn = 1.0. When F⁻ anion substitutes an O2- anion, free electrons will be generated for charge compensation to result in the increased n with increasing F/Sn from 0 to 0.5. However, there is a certain saturation limit that excess F⁻ cannot substitute O2⁻ anymore, and the F⁻ anions may be expelled to segregate on grain boundaries. This results in the slight decrease of n at F/Sn = 1. The present behavior of n vs. F-doping is consistent with those of the FTO films prepared by magnetron sputtering [n variation, the μ decreased slightly from 7.6 to 4.5 cm2/Vs when F/Sn increased from 0 to 0.5, and then it increased to 7.7 cm2/Vs at F/Sn = 1. The electrical properties  is described by Matthiessen’s rule [impurityμ, gbμ, phononμ, and hoppingμ are factors that influence mobility from impurity scattering, grain boundary scattering, phonon scattering, and retardation by hopping transport, respectively. In TCO thin films, the ionized impurities and grain boundaries are found to be the main scattering mechanisms [L) of thin films can be estimated by the equation L values of the present FTO films are in range of 0.9–1.7 nm, which is far smaller than grain size; thus, grain boundary scattering is not the dominant scattering mechanism. A higher n usually creates more scattering centers that lead to reduced μ, as shown in n and μ, the resistivity monotonically decreased from 4.1 to 0.7 mΩcm with increasing F/Sn from 0 to 1, as 1/ρ = nμ|e|, where e is the electron charge. This F-doping-dependent ρ is consistent with that for the FTO films [The mobility  of the 286–317 nm-thick films were 91.1, 90.4, 90.2, and 84.4% for F/Sn ratios of 0, 0.1, 0.5, and 1, respectively. This means that the average transmittance remains above 90% for F/Sn ≤ 0.5, which is higher than the value of 80% for the FTO prepared by APCVD [aR = 20.7 nm and defect concentration. The present F-doping-dependent transmittance is consistent with the result for the FTO films prepared by the sol-gel process [As shown in by APCVD . Moreove process . ρ of 0.7 mΩcm for the film prepared at F/Sn = 1.0 in this study is considerably higher than those of the FTO films with ρ in range of 1.5–6.5 mΩcm [ρ values of the FTO films [6.5 mΩcm ,18,19, bTO films ,18. In aTO films ,13,17.P–h) curves of FTO thin films prepared at different F/Sn ratios from 0 to 1, which provides the information about elastic behavior and plastic deformation. From the P–h data, we employed the Oliver and Pharr method to calculate the hardness (H) and Young’s modulus , namely, S) is determined as S = dP/dh, i.e., the slope of the initial portion of the unloading curve. The contact depth can be estimated from the P–h data using ε is the indenter constant (0.75 for a Berkovich indenter tip) and β is a geometric constant (β ≈ 1 for a Berkovich indenter tip). The reduced elastic modulus (rE) is determined as follows:Particularly, the hardness was estimated as erkovich . The conrelation  S=2βErApv is the Poisson’s ratio and i and f denote parameters for the indenter and FTO thin films, respectively. The elastic modulus  and Young’s modulus–displacement (E–h) curves of the films prepared at different F/Sn ratios. The H–h and E–h curves can be divided into two stages, i.e., (1) the H or E gradually increases to reach a maximum value as h is increased and (2) subsequently decreases to an almost constant value. The first stage is usually attributed to the transition from purely elastic to elastic/plastic contact, and the hardness is not accurately measured by the mean contact pressure at this stage. Instead, the mean contact pressure can represent the hardness only under the condition of a fully developed plastic zone. After the first stage, the H and E reach the constant values, which are regarded as intrinsic properties of the films. As shown in H and E values were in a reasonable range compared with the previous studies [2 film (F/Sn = 0) was comparable with the H = 6.1 ± 0.1 GPa and E = 78.5 ± 0.4 GPa of SnO2 films prepared by spray-pyrolysis [H of 6.9–10.2 GPa and E of 115.1–131.7 GPa for the present FTO films were larger than H = 5.1 GPa and E = 71.1 GPa for FTO thin films deposited by chemical vapor deposition [H = 9.01 GPa and E = 125.24 GPa for the as-deposited FTO film prepared by APCVD [ studies ,16,43. IH, E, and D of the FTO thin films. The H and E results are mostly determined by the D value of FTO thin films, and follow an inverse Hall-Petch effect. A larger D leads to the larger H and E values. For examples, when D increased from 29.6 to 42.7 nm and then decreased to 35.1 nm, the corresponding H increased from 5.6 ± 0.2 to 12.3 ± 0.4 GPa and then decreased to 10.2 ± 0.3 GPa. It is worthy to note that the inverse Hall-Petch effect occurs in nanoscale granular materials with grain size below a certain critical value, for example 10 nm [H, E vs. D behavior agrees well with that of FTO thin films grown by APCVD [H and E values decrease when increasing the level of porosity of the materials [2 film (F/Sn = 0), the surface morphology with spear-like particles may have internal voided structure near the surface due to the shadowing effect during the thin film growth [H and E values for the SnO2 film compared to those of FTO films. le 10 nm , and it le 10 nm ,45,46. Rby APCVD . In addiaterials . For them growth ,49. This3Se2Te films  [H/E > 0.1 values of some brittle materials, such as ceramic Mo-incorporation β-Ga2O3 films [fH/E = 0.093 and The plasticity index (n energy . Meanwhipagation . The H/E59–0.093 . These H8–0.031) , and reaO3 films  and hardO3 films . In addiain size . The FTO2 with dominant preferred orientation of (110), and the grain size tended to increase as the F/Sn ratio increased. The films had granular surface morphologies with spear-like particles for F/Sn = 0 and coconut shell-like particles for F/Sn = 0.1, 0.5, and 1.0, and they were compact and possessed good continuity. XPS results confirmed the presence of F-doping with concentrations of few percent . By increasing F/Sn ratios, the films achieved significant reduction in resistivity from 4.1 mΩcm at F/Sn = 0 to 0.7 mΩcm at F/Sn = 1, primarily due to an order increase of n for F/Sn = 0.5 and 1.0. The films achieved excellent optical property, with an average transmittance in the visible range over 90% for F/Sn of 0–0.5. Importantly, the H and E of the FTO films were remarkably enhanced by introducing F-doping, following the inverse Hall-Petch effect. The highest H and E values were 12.3 ± 0.4 GPa and 131.7 ± 8.0 GPa for the FTO films prepared at F/Sn of 0.5 (with 5.1% F and aR of 11.9 nm). The FTO thin films prepared at F/Sn = 0.5 also possessed the largest plasticity index (fH/E = 0.093) and plastic deformation resistance (FTO thin films were successfully grown on glass substrates using USP. Effects of F/Sn atomic ratio on structural, morphological, compositional, electrical, optical, and nanomechanical properties of the films were studied. The films exhibited tetragonal rutile structure of SnO


During early developmental windows known as critical periods (CPs) of plasticity, passive alterations in the quality and quantity of sensory inputs are sufficient to induce profound and long-lasting distortions in cortical sensory representations. With CP closure, those representations are stabilized, a process requiring the maturation of inhibitory networks and the maintenance of sufficient GABAergic tone in the cortex. In humans and rodents, however, cortical inhibition progressively decreases with advancing age, raising the possibility that the regulation of plasticity could be altered in older individuals. Here we tested the hypothesis that aging results in a destabilization of sensory representations and maladaptive dysregulated plasticity in the rat primary auditory cortex (A1). Consistent with this idea, we found that passive tone exposure is sufficient to distort frequency tuning in the A1 of older but not younger adult rats. However, we also found that these passive distortions decayed rapidly, indicating an ongoing instability of A1 tuning in the aging cortex. These changes were associated with a decrease in GABA neurotransmitter concentration and a reduction in parvalbumin and perineuronal net expression in the cortex. Finally, we show that artificially increasing GABA tone in the aging A1 is sufficient to restore representational stability and improve the retention of learning. In this study, we examined brain plasticity in the auditory cortex of young adult and older adult rats in the context of different types of auditory stimulation and training. Surprisingly, older brains retained an equal or even higher potential for plasticity compared to young adults. In older brains, however, changes elicited by auditory stimulation and training were rapidly lost, suggesting that such increased plasticity might be detrimental, as the older brains were unable to consolidate these changes. This increased but poorly regulated plasticity was associated with a reduction in cortical inhibition, which normally maintains the stability of sensory representations in the young adult brain. Importantly, increasing inhibition artificially with clinically available drugs restored stability and improved the retention of learning.+) interneurons  of plasticity, passive exposure to environmental sounds profoundly shapes frequency tuning in the rat primary auditory cortex A1; . On closrneurons  and perirneurons . In the rneurons  or disrurneurons . Plasticrneurons . This tirneurons .+ and somatostatin-positive (SST+) cells in older brains  to P24], 24 Long-Evans young adult rats of either sex (age 6–8 mo), and 28 Long-Evans old adult rats of either sex (22–24 mo) were used for this study.The tone pip-exposed rats were housed for 1 or 2 consecutive weeks  in a sound attenuated chamber equipped with a speaker. The exposure sequences were generated using custom Matlab routines and contained repetitive trains of six 25-ms-long 5- or 10-kHz pips with 5-ms cosine gates presented at a rate of 5 p.p.s. at an intensity of 70 dB SPL.For A1 mapping, the rats were premedicated with dexamethasone (0.2 mg/kg) to minimize brain edema. They were anesthetized with ketamine/xylazine/acepromazine  followed by a continuous delivery of isoflurane 1% in oxygen delivered via endotracheal intubation and mechanical ventilation. Vital signs were monitored using a MouseOx device (Starr Life Sciences). Body temperature was monitored with a rectal probe and maintained at 37°C with a homeothermic blanket system. The rats were held by the orbits in a custom-designed head holder, leaving the ears unobstructed. The cisterna magna was drained of cerebrospinal fluid to further minimize brain edema. The left temporalis muscle was reflected, auditory cortex (AC) was exposed, and the dura was resected. The cortex was maintained under a thin layer of silicone oil to prevent desiccation.Cortical responses were recorded with 32–64-channel tungsten microelectrode arrays (Neuronexus). The microelectrode array was lowered orthogonally into the cortex to a depth of 470–600 µm (layers 4/5) where vigorous stimulus-driven responses were obtained. The extracellular neural action potentials were amplified, filtered (0.3–5 kHz), sorted, and monitored on-line. Acoustic stimuli were generated using TDT System III (Tucker-Davis Technologies) and delivered in a free-field manner to the right ear through a calibrated speaker (Tucker-Davis Technologies). A software package  was used to generate acoustic stimuli, monitor cortical response properties on-line, and store data for off-line analysis. The evoked spikes of a single neuron or a small cluster of neurons were collected at each site.Frequency-intensity receptive fields were reconstructed by presenting pure tones of 63 frequencies  at 8 sound intensities (0–70 dB SPL in 10-dB increments) to the contralateral ear at a rate of one stimulus per second. Ten-minute-long trains of 50-ms tone pips were presented at 3 pulses per second at a sound intensity of 70 dB SPL. Each train had a commonly occurring frequency (standard) with a probability of occurrence of 80% and 5 pseudo-randomly distributed oddball frequencies presented 20% of the time with no repetition. The oddball frequencies in the train had a constant separation of 1 octave.The characteristic frequency (CF) of a cortical site was defined as the frequency at the tip of the V-shaped tuning curve. For flat-peaked tuning curves, the CF was defined as the midpoint of the plateau at threshold. For tuning curves with multiple peaks, the CF was defined as the frequency at the most sensitive tip . The CF and threshold were determined using an automated routine developed in the Matlab environment (The MathWorks).To generate A1 maps, Voronoi tessellation (a Matlab routine) was performed to create tessellated polygons with electrode penetration sites at their centers. Each polygon was assigned the characteristics  of the corresponding penetration site. In this way, every point on the surface of the AC was linked to the characteristics experimentally derived from its closest sampled cortical site. Primary AC (A1) was identified based on its rostral-to-caudal tonotopy, reliable short-latency tone-evoked neuronal responses, and relatively sharp V-shaped RF. To examine A1 map plasticity, we compared the percentage of A1 sites with CFs in 12 bins (width = ½ octave) spanning the spectrum of presented tones.Normalized responses to standard and oddball tones were obtained by dividing the average firing rate recorded in the 50 ms after the occurrence of each tone presentation by the average firing rate observed during the 50 ms after the first standard or oddball tone in the sequence. Asymptotes for standard and oddball responses were calculated by fitting exponential functions with a least squares method to the normalized response data from each recorded neuron. Simple linear regression of the normalized responses to the standard tone for the interval from event no. 150 to event no. 1200 was performed. The slope of the resulting best fit line was computed to determine the level of adaptation for each recorded site.Behavior was shaped in three phases. During the first phase, rats were trained to make a nose poke response to obtain a food reward. During the second phase, rats were trained to make a nose poke only after presentation of an auditory stimulus. During the third phase, the actual training program, rats were trained to make a nose poke only for the target stimulus (a 5-kHz pure tone) and not for a foil nontarget stimulus (10-kHz pure tone). The tones were presented at 60 dB SPL, stimulus presentation was randomized, and the probability of a target stimulus presentation was set at 20%. Training was performed in an acoustically transparent operant training chamber  contained within a sound-attenuated chamber. Sound presentation and response recording were performed using OpenEx software and RZ6 auditory processing hardware from Tucker-Davis Technology and delivered in a free field manner through a calibrated loudspeaker.The intertrial interval was selected at random from a range of 4–6 s. A rat’s behavioral state at any point in time was classified as either “go” (producing a nose poke behavior) or “no-go.” For a given trial, the rat could elicit one of four reinforcements produced by the combinations of responses (go or no-go) and stimulus properties (target or nontarget). Go responses within 5 s of a target were scored as a hit; a failure to respond within this time window was scored as a miss; a go response within 5 s of a nontarget stimulus was scored as a false positive; the absence of a response was scored as a withhold. A hit triggered the delivery of a food pellet. A miss or false positive initiated a 5-s time-out period during which time the house lights were turned off and no stimuli were presented. A withhold did not produce a reward or a time-out. Psychometric functions and stimulus target recognition indexes (D-prime) were calculated for each training session by plotting the percentage of go responses as a function of the total number of target stimuli  and the percentage of false positives as a function of the total number of foils . Learning curves were reconstructed by plotting the D-prime measure reached over successive days of training.Immediately after craniotomy (see Methods: Electrophysiology), a microdialysis probe  was implanted in the AC using the stereotaxic coordinates : bregma m PBS at pH 7.2. Immediately after perfusion, rat brains were removed and placed in the same fixative overnight for further fixation and then transferred to a 30% sucrose solution, snap-frozen, and stored at –80°C until sectioning. Fixed material was cut in the coronal plane along the tonotopic axis of A1 on a freezing microtome at 40 µm. Tissue was incubated overnight at 4°C in either monoclonal or polyclonal antisera . Tissue samples were always processed in pairs during immunostaining procedures to limit variables relative to antibody penetration, incubation time, and post-sectioning age/condition of tissue. A Zeiss LSM 510 Meta confocal microscope was used to assess fluorescence in the immunostained sections. Quantification of PV+ cells and PNN optical density was performed in ImageJ  and MetaMorph imaging software (Molecular Devices Systems), respectively. Digital images of A1 cortical sections were taken with a 40× objective (Zeiss LSM 510). All quantification was assessed in 300–400-µm-wide A1 sectors  extending from layer 1 to the underlying white matter by an experimenter blind to the age of the animals. PV+ cells were classified into four subclasses as follows: low-PV, 0–0.8 × 105; intermediate low-PV, 8–1.6 × 105; intermediate high-PV, 1.6–2.4 × 105; high-PV, >2.4 × 105. PNNs were classified into four subclasses as follows: low PV, 0–1 × 104, intermediate low-PV, 1–2 × 105; intermediate high-PV, 2–3 × 105; high-PV, >3 × 105.Immediately following the end of recording sessions, rats received a high dose of pentobarbital (85 mg/kg i.p.) and were perfused intracardially with 4% paraformaldehyde in 0.1 t tests or two-way analysis of variance with Tukey post hoc correction for multiple comparisons. Wilcoxon rank-sum test or Kruskal–Wallis test with Tukey post hoc correction for multiple comparisons were used for nonparametric data analysis. Data are presented as mean ± SEM or median ± median absolute deviation (MAD). Superscript letters listed with p-values correspond to the statistical tests shown in For normally distributed data, statistical significance was assessed using unpaired two-tailed n = 4) to 5 kHz tone pips for 1 week. For comparison, the same exposure was used in young adult  and immature rats in their CP window  = 14.84, p < 0.001a). Such an exposure resulted in a significant overrepresentation of the exposure tone in A1 of immature rats  but not in the young adult group  = 4.02, p < 0.001; 3.84 ± 1.3% increase, p = 0.87b, with Tukey–Kramer correction; F = 10.77, p < 0.001, two-way ANOVA; 8.05 ± 1.14% increase, p < 0.001c, with Tukey–Kramer correction).Aging is characterized by a progressive reduction in cortical inhibition to levels akin to those observed during developmental critical periods . This ran = 4) and old (n = 4) adult rats were exposed to 10-kHz tone pips during the first week and to 5-kHz tone pips during the second week  = 13.13, p < 0.001, two-way ANOVA; 5 kHz: 8.81 ± 1.7% increase, p < 0.001; 10 kHz: 5.18 ± 1.3% decrease, p = 0.35d, with Tukey–Kramer correction) but did not alter the frequency tuning map of young adults  = 2.69, p = 0.005, two-way ANOVA; 5 kHz: 2.1 ± 0.56% decrease, p = 1; 10 kHz: 3.4 ± 0.57% decrease, p = 0.96e, with Tukey–Kramer correction; To further document the extent of this tuning instability in older rats, we examined the effect of two consecutive pure-tone exposures over a 2-week period. Young  or diazepam (right) intraperitoneal injections during the 2-week passive exposure period. B, Difference in frequency tuning between naïve and treated rats expressed as A1 percentage area and separated by CF. Values shown are mean ± SEM; two-way ANOVA corrected for multiple comparisons using Tukey–Kramer test. Download Figure 2-1, EPS file.Immature rats exposed sequentially to pure tones over 2 weeks show an overrepresentation of the first tone of exposure. Rats were exposed starting at P10 to 5-kHz pure tones for 1 week, followed immediately by exposure to 10-kHz pure tones for 1 week. A agonist diazepam  during two consecutive pure-tone exposures as described above  = 7.23, p < 0.001, two-way ANOVA; 5 kHz: 2.23 ± 1.45% increase, p = 1; 10 kHz: 13.8 ± 5.7% increase, p < 0.001f, corrected with Tukey–Kramer test; Having documented the existence of age-related tuning instability in A1, and considering the reduction of intracortical inhibition in sensory cortices associated with advanced age , we hypon = 5). Previous experiments have shown that passive tone exposure outside the CP for frequency tuning (∼P10–P14) does not alter the A1 tonotopic map  = 14.62, p = 0, two-way ANOVA; 5 kHz: 13.27 ± 3.4% increase, p < 0.001; 10 kHz: 5.72 ± 1.6% decrease, p = 0.15g, corrected with Tukey–Kramer test; To investigate whether sequential exposure to pure tones would have a similar effect in immature animals as in old adult animals, we used a sequential 2-week exposure paradigm starting at P10  = 12.58, p < 0.001, two-way ANOVA; 5 kHz: 11.93 ± 3.6% increase, p = 0.018; 10 kHz: 6.11 ± 1.1% decrease, p = 0.1h, corrected with Tukey–Kramer test; Although treatment with diazepam accelerates the closing of the CP , it doesAuditory neurons continuously monitor the environment, suppressing their response to repetitive sounds and making novel stimuli more salient . In the 10.1523/ENEURO.0051-18.2018.f3-1Figure 3-1A, Cumulative distribution plot of responses to repetitive tones  for all experimental groups  in immature (I) and old adult rats relative to young adults (YA). Adaptation was restored with the local administration of midazolam  = 10.21; YA: 0.31 ± 0.019; I: 0.44 ± 0.017, p = 1.24 × 10–5, relative to YA; I-MDZ: 0.28 ± 0.015, p = 1.20 × 10–5, relative to I; OA: 0.43 ± 0.032, p = 5.9 × 10–4, relative to YA; OA-MDZ: 0.34 ± 0.027, p = 0.018, relative to OA). No significant differences in the overall magnitude of responses to oddballs (circles) was found between groups  = 1.24). Both immature and aged groups showed a diminished response gap between standards and oddballs . This gap improved with the local administration of midazolam for immature but not old adult rats  = 3.84; YA, 0.30 ± 0.033; I, 0.19 ± 0.026, p = 0.057, relative to YA; OA, 0.15 ± 0.035, p = 0.0172, relative to YA; I-MDZ, 0.32 ± 0.024, p = 0.015 relative to I; OA-MDZ, 0.19 ± 0.041, p = 0.92, relative to OA; corrected for multiple comparisons). Immature group: n = 8, recorded sites = 376; YA: n = 4, recorded sites = 205; OA: n = 4, recorded sites = 192. YA group: n = 4, recorded sites = 205; I: n = 8, recorded sites = 376; OA: n = 4, recorded sites = 192; I-MDZ: n = 8, recorded sites = 346; OA-MDZ: n = 4, recorded sites = 155. Values shown are mean ± SEM. *p < 0.05. Download Figure 3-1, EPS file.Summary of adaptation in response to repetitive tones. oups see B. B, Red−4, number of recorded cortical sites = 205) but an increase in responses to repetitive tones in the immature and old adult groups relative to YA  = 5.64k, relative to YA; OA, 0.15 ± 0.09 p = 9 × 10−4, t(395) = 3.35l, relative to YA; t test). Interestingly, the sum of responses to high and low probability tones remained constant in the immature and young adult groups, while it increased for the old adult group  = 0.75m; YA, 4.78 ± 5, p = 0.52, t(408) = 0.64n; OA, 13.83 ± 2.71, p = 0.011, t(383) = 2.55o; paired t test; Tuning stability in the same A1 neurons was examined by interspersing five oddball (low-probability) tones covering the hearing range during the repetitive (high-probability) tone presentation (see Methods). Using this method, coarse tuning curves could be constructed over two time intervals during the tone train exposure . On average, A1 neurons in the immature and old adult groups exhibited a significant increase in response to the high-probability tone from T1 to T2, while the opposite was seen in the young adult group  and passive exposure (1–2 weeks) experiments, we decided to use midazolam as opposed to the long-acting GABAA agonist diazepam. Direct application of midazolam (1 μg/µl at a rate of 0.5 μl/min) to the cortex resulted on average in the progressive suppression of A1 responses to repetitive tones in the immature and older groups . It also resulted in a significant decrease in response to the high-probability tone in these groups  = 5.29r; OA: –0.09 ± 0.06, p = 0.03, t(345) = 2.1s; t test). The overall response to the standard-oddball stimulus remained constant from T1 to T2 for both groups  = 0.86t; OA, 1.63 ± 5.52, p = 0.94, t(308) = 0.08u; t test; B.In immature rats, short periods of auditory stimulation readily modify frequency tuning in A1, likely because of a disrupted E/I balance following the onset of hearing . Additioates SSA , we hypon = 8) and older (n = 12) adult rats on an auditory discrimination task and then measured training-induced A1 changes at the end of training and after a 4-week delay. Both groups were trained on a two-tone discrimination task . Training ended once the rats’ discrimination reached a sustained value of D-prime (d′) ≥1 for two consecutive days; p = 0.032, t(18) = 2.32v; t test; n = 4) and a subgroup of old adult rats  = 13.42, p < 0.001w; F = 6.57, p < 0.001x; respectively). Compared to age-matched controls, both groups exhibited an increase in the number of neurons tuned to the target tone by the end of the training period  and older  rats after a 4-week delay period following completion of training  = 5.02y; delay: F = 6.68, p < 0.001, two-way ANOVA; 5 kHz: 10.37 ± 2.3% increase, p = 0.01z, corrected with Tukey–Kramer test; OA-Tdelay: F = 1.41, p = 0.18ab, two-way ANOVA; 5 kHz: 4.72 ± 2.1% increase; n = 4) of older rats with diazepam  during the delay period post-training. A1 mapping in this group revealed a persistent target tone overrepresentation not significantly different from what had been observed immediately following training  = 5.42, p < 0.001, two-way ANOVA; 5 kHz: 7.64 ± 2.8% increase, p = 0.022ac, corrected with Tukey–Kramer test; To determine the retention of learning and persistence of training-related A1 retuning, we characterized trained younger  than in young adult controls  when measured in silence  = 2.53ad, t test; p = 0.01, t(6) = 3.66ae, t test; To study the anatomic correlates of frequency tuning instability and impaired training performance, we sampled GABA concentration using microdialysis and quantified PV/PNN expression in A1 through immunohistochemistry. As documented in previous research , we foun+ neurons and associated PNNs are important regulators of experience-dependent plasticity throughout life  × 105; H(4) = 14.52, p = 0.0058, Kruskal–Wallis test; I: 1.17 ± 0.85, p = 0.52, relative to YA; YA: 1.19 ± 0.69; OA: 1 ± 0.53, p = 0.011, relative to YA; ID: 1.3 ± 0.66, p = 0.96, relative to YA; OAD: 1.1 ± 0.76, p = 0.97af, relative to YA; Tukey–Kramer test; + cells, diazepam treatment resulted in recovery of PNN intensity staining for immature and older adult rats  = 83.97, p < 0.0001, Kruskal–Wallis test; I: 0.7 ± 0.79, p < 0.0001, relative to YA; YA: 1.64 ± 1.37; OA: 0.76 ± 1.04, p < 0.001, relative to YA; ID: 1.79 ± 1.39, p = 0.96, relative to YA; OAD: 1.11 ± 1.33, p = 0.003ag, relative to YA; Tukey–Kramer test; To assess whether age-related representational instability would be paralleled by a reduction in these plasticity-regulating structural elements, we first characterized the expression of the main interneuron subpopulations in the context of total cell counts for the three age groups—immature, young adult, and older adult—included in the present study  during silence and (B) during auditory stimulation from young adult  and old adult  rats. Values shown are mean ± SEM. *p < 0.05, **p < 0.01, t test. Download Figure 5-1, EPS file.GABA concentration is reduced in the old adult A1. GABA concentration in A1 dialysate obtained , 6 months (n = 6), and 24 months (n = 6). Download Figure 5-2, DOCX file.Interneuron cell count in A1 across the lifespan of the rat. Number of PV-, SST-, PNN-, GABA-, and Nissl-positive cells per field at P15  PV+ and (B) SST+ immunolabeled cells costained for GABA at the age intervals defined in Fig. 5-2. Download Figure 5-3, EPS file.PV and SST expression in A1 interneurons. Representative high power confocal micrographs of  Cumulative distribution plot and (B) individual variability of PV-labeling intensity for all experimental groups (see C) Cumulative distribution plot and (D) individual variability of PNN-labeling intensity for all experimental groups . Download Figure 5-4, EPS file.Restoration of PVoups see B. (C) Cuoups see D. Note t+ cells could be divided into four subgroups: low, intermediate low, intermediate high, and high intensity (+ cells in older rats compared to young adults (H(4) = 13, p = 0.011aj, Kruskal–Wallis test; fraction of PV+ cells with low staining intensity and p-value relative to YA, per group: Ilow = 16 ± 7.1%, p = 0.82; YAlow = 14 ± 3.2%; OAlow = 2.9 ± 0.4%, p = 0.005; IDlow = 12.1 ± 1.7%, p = 0.99; OADlow = 14.2 ± 3.2%, p = 0.99ah; Tukey–Kramer test; H(4) = 17.24, p = 0.0017, Kruskal–Wallis test; fraction of low-intensity PNNs and p-value relative to YA, per group: Ilow = 55 ± 8.1%, p = 0.48; YAlow = 34 ± 2.2%; OAlow = 55 ± 1.7%, p = 0.04ai, Tukey–Kramer test) and a decrease in the high-intensity PNN subgroup in immature and aged rats compared to young adults (H(4) = 22.06, p < 0.001, Kruskal–Wallis test; fraction of high-intensity PNNs and p-value relative to YA, per group: Ihigh = 3.5 ± 1.7%, p = 0.004; YAhigh = 26 ± 1.9%; OAhigh = 9.3 ± 2.7%, p = 0.039aj; Tukey–Kramer test). Notably, following diazepam treatment, the proportion of low-intensity PNNs in older rats decreased, whereas the proportion of high-intensity PNNs in both immature and older rats increased, resulting in an intensity staining distribution that resembled that of the control (YA) group  are exploring the effects of manipulating cortical E/I balance on learning in the elderly . For insTraditionally, aging has been regarded as a period of limited plasticity. However, our experiments suggest that this idea is unlikely to be correct in detail, as the aged brain is in some ways more plastic than the young adult brain. We propose that the inhibitory regulation of plasticity, rather than plasticity per se, is reduced in the aged brain . Researc


Upon noggin/Dkk-1/IGF-1 induction, the treated ASCs showed elevated expression of retinal progenitor, retinal ganglion, and photoreceptor cell markers as well as the glutamate-evoked calcium response, which was not observed in the noninduced cells. Compared to the regular induction treatment, Notch signaling activation by JAG1 enhanced the expression of retinal progenitor and precursor markers without affecting the glutamate-evoked calcium response. In contrast, Notch signaling inhibition by DAPT showed more retinal ganglion cells, but delayed the response to glutamate stimulation. In summary, our results revealed that human ASCs possess a retinal transdifferentiation potential upon noggin/Dkk-1/IGF-1 induction, which can further be enhanced by Notch signaling activation.Retinal disease treatment by stem cell-based replacement relies on stem cell differentiation into retinal cells. We previously demonstrated that human periodontal ligament-derived stem cells can be directed into retinal lineage upon induction. Here, we report the transdifferentiation potential of human adipose-derived stem cells (ASCs) into retinal lineage and its enhancement by Notch signaling modulation. Human ASCs, isolated from abdominal fat, expressed mesenchymal but not hematopoietic stem cell markers, and they can differentiate into adipocytes, chondrocytes, and osteoblasts  Retinal diseases, including age-related macular degeneration, glaucoma, diabetic retinopathy, and retinitis pigmentosa, are the leading causes of irreversible blindness and visual impairment, affecting more than 300 million people worldwide. The human retina has limited intrinsic regenerative capability, and current treatment regimens are not sufficient for functional repair of the diseased retinal cells. Stem cell-based replacement, which relies on the differentiation of stem cells into retinal cells, could be a potential strategy for retinal disease treatment , 2.Retinal progenitor cells (RPCs), embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs) have been intensively studied for retinal cell synthesis , 4. Yet,in vitro with ectopic expression of paired box protein-6 (PAX6) gene in a fibronectin-supplemented medium i = KR/{K/([Ca2+]rest + 1) − R}, where K is the dissociation constant of Fluo-4 AM (400 nM), R is the fluorescence ratio (ΔF/Fbaseline), and [Ca2+]rest is the resting Ca2+ concentration .Spontaneous intracellular calcium transient was evaluated using fluo-4-acetoxymethyl ester . Briefly, the treated or control human ASCs at Day 24 were incubated in Hanks' balanced salt solution  with post hoc Tukey's test were used to compare the means among the treatment groups. Significance was defined as p < 0.05.The data were presented as mean ± standard deviation (SD) from the 3 independent cell lines. All statistical analyses were performed using a commercially available software . Independent Human ASCs were first characterized by the expression of MSC  and HSC  markers. Flow cytometry analysis showed that CD44 expression was found in 99.10 ± 1.01%, CD73 in 97.07 ± 2.56%, CD90 in 99.90 ± 0.24%, and CD105 in 99.45 ± 0.39% of human ASCs . In contHuman ASCs were treated with retinal induction medium for 24 days . A spherPAX6 and NES), photoreceptor , and RGC (ATOH7 and TUBB3) marker genes time-dependently increased along the retinal induction treatment period (PAX6 and NES genes were increased in the treated human ASCs by 38.03 ± 8.41-fold (p < 0.05) and 38.41 ± 4.12-fold (p < 0.01), respectively, compared to the expression at Day 0. Similarly, CRX, NRL, RHO, and RCVRN genes were increased by 15.46 ± 1.75-fold (p < 0.05), 3.94 ± 0.81-fold (p < 0.05), 14.25 ± 1.96-fold (p < 0.05), and 245.90 ± 15.46-fold (p < 0.05), respectively. Meanwhile, the expression of ATOH7 and TUBB3 genes in the retinal-induced ASCs at Day 24 were increased by 16.65 ± 3.35-fold (p < 0.05) and 1.75 ± 0.19-fold (p < 0.05), respectively. In contrast, all retinal marker genes showed no significant change in the negative control group throughout the treatment period.To verify the retinal differentiation capacity of human ASCs, the expression of retinal lineage markers was examined at Days 0, 10, 17, and 24 after induction. Gene expression analysis showed that the expression of RPC (t period . Sybr grp < 0.01) and 3.00 ± 0.53-fold increase , CRX , POU4F2 , and TUBB3  observed in the retinal-induced ASCs were 525.18 ± 228.80%, compared to 133.79 ± 159.82% in the negative control group (p < 0.01).The function of the retinal-induced ASCs was evaluated by the glutamate-evoked calcium response. A robust increase in fluorescence intensities was observed in the retinal-induced ASCs after glutamate stimulation, but only a very weak signal was observed in the negative control group . The fluTo confirm the specificity of retinal transdifferentiation, the retinal-induced ASCs were evaluated with the staining for adipogenesis and osteogenesis, and negative staining of lipid deposition, alkaline phosphatase, and calcium deposition was observed in the retinal-induced ASCs (Supplementary HES1 gene). In the JAG1 treatment group, the HES1 gene was significantly upregulated by 1.50-fold at Day 24 . Notably, the expression of the Notch signaling receptor (NOTCH1 gene) was also upregulated in the JAG1 treatment group by 1.76-fold (p < 0.01), but not changed in the DAPT treatment group.To delineate the regulation of Notch signaling in the retinal differentiation of human ASCs, JAG1  and DAPT  were applied to the retinal induction medium . The effPAX6 gene was upregulated in the JAG1 treatment group by 1.93 ± 0.31-fold at Day 24 (p < 0.001), compared to the regular induction group. Similarly, the expression of the NES gene was also increased in the JAG1 treatment group by 1.37 ± 0.21-fold at Day 24 (p < 0.05). In contrast, the expression of the NES gene was downregulated in the DAPT treatment group by 1.32 ± 0.22-fold (p < 0.05). For the photoreceptor markers, the expressions of CRX, NRL, and RHO genes were upregulated by 1.51 ± 0.28-fold (p < 0.01), 1.40 ± 0.17-fold (p < 0.01), and 2.55 ± 1.15-fold (p < 0.001), respectively, in the JAG1 treatment group at Day 24, compared to those in the regular induction group. For the RGC markers, the expressions of the ATOH7 and TUBB3 genes were increased in the JAG1 treatment group by 1.78 ± 0.31-fold (p < 0.01) and 1.15 ± 0.06-fold (p < 0.05), respectively, at Day 24, compared to the regular induction group. Human ASCs in the DAPT treatment group showed a similar retinal marker gene expression to that in the regular induction treatment group.For the gene expression analysis on RPC markers, the expression of the p < 0.05), compared to the regular induction group (p < 0.01) at Day 24, compared to the regular induction group. Furthermore, the percentage of PAX6 nucleus-positive cells in the JAG1 treatment group  was higher than that in the regular induction group  was significantly higher than that in the regular induction group (40.72 ± 7.01%), and the percentage of CRX nuclear-positive cells was also significantly higher in DAPT treatment group . The percentages of TUBB3-positive cells were lower in the JAG1 treatment group , but higher in the DAPT treatment group , compared to that in the regular induction group (21.25 ± 6.18%). In addition, the percentage of POU4F2 nuclear-positive cells was higher in the DAPT treatment group  than that in the regular induction group (35.10 ± 7.86%).Coherent to the gene expression results, the immunoblotting analysis showed that the expression of the PAX6 protein was significantly upregulated in the JAG1 treatment group by 1.45 ± 0.33-fold at Day 24 (on group . Similarp < 0.05). Besides, the time response to the glutamate stimulation in the DAPT-treated cells was also significantly delayed , photoreceptor markers , and neuronal and retinal ganglion cell markers . Moreover, the induced PDLSCs showed a significant response to excitatory glutamate [PAX6 and NES), photoreceptor , and RGC (ATOH7 and TUBB3) markers were significantly upregulated during the induction treatment period in a time-dependent manner  and its downregulation in DAPT treatment  as well as mature photoreceptor (RHO) and RGC (TUBB3) markers (Figures NES gene, but showed more POU4F2- and TUBB3-positive cells, suggesting that Notch signaling inhibition would reduce the RPC population but increase the production towards the retinal ganglion lineage. Yet, DAPT treatment also delayed the response of glutamate stimulation in the retinal-induced ASCs (In this study, we confirmed the efficacy of Notch signaling modulation with the upregulation of  Figures . JAG1 tr Figures , indicatced ASCs , suggestin vitro disease modeling, and stem cell replacement therapy in the future.In summary, human ASCs possess the retinal differentiation potential upon noggin/Dkk-1/IGF-1 induction treatment, and the synthesis of retinal progenitor and precursor cells can further be enhanced by Notch signaling activation. Human ASCs should be a promising autologous source of adult stem cells for retinal cell synthesis,