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Chaperone-mediated autophagy plays an important role in regulating retinal progenitor cell homeostasis
Stem Cell Research & Therapy volume 13, Article number: 136 (2022)
To explore the function and regulatory mechanism of IFITM3 in mouse neural retinal progenitor cells (mNRPCs), which was found to be very important not only in the development of the retina in embryos but also in NRPCs after birth.
Published single-cell sequencing data were used to analyze IFITM3 expression in mNRPCs. RNA interference was used to knock down the expression of IFITM3. CCK-8 assays were used to analyze cell viability. RNA-seq was used to assess mRNA expression, as confirmed by real-time quantitative PCR, and immunofluorescence assays and western blots were used to validate the levels of relative proteins, and autophagy flux assay. Lysosomal trackers were used to track the organelle changes.
The results of single-cell sequencing data showed that IFITM3 is highly expressed in the embryo, and after birth, RNA-seq showed high IFITM3 expression in mNRPCs. Proliferation and cell viability were greatly reduced after IFITM3 was knocked down. The cell membrane system and lysosomes were dramatically changed, and lysosomes were activated and evidently agglomerated in RAMP-treated cells. The expression of LAMP1 was significantly increased with lysosome agglomeration after treatment with rapamycin (RAMP). Further detection showed that SQSTM1/P62, HSC70 and LAMP-2A were upregulated, while no significant difference in LC3A/B expression was observed; no autophagic flux was generated.
IFITM3 regulates mNRPC viability and proliferation mainly through chaperone-mediated autophagy (CMA) but not macroautophagy (MA). IFITM3 plays a significant role in maintaining the homeostasis of progenitor cell self-renewal by sustaining low-level activation of CMA to eliminate deleterious factors in cells.
Retinal progenitor cells (RPCs) have been investigated for years, and their transplantation or endogenous activation represent promising potential therapeutic avenues in eye disease in the future [1, 2]. Therefore, investigation into the effective amplification of these cells in vitro for a long period [3] and the mechanisms by which they are regulated are particularly important. Here, we analyzed the published dataset of the developing murine retina of single-cell RNA sequencing, and the results suggested that interferon (IFN)-induced transmembrane protein 3 (IFITM3) was highly expressed not only in the development of the retina in embryos but also in the retina after birth [4].
IFITM3 is a transmembrane protein that localizes to several cellular components, including the apical part of the cell, cell surface and endoplasmic reticulum (ER), and is well-known for its role in modulating the interferon-mediated innate immune system to defend against invading pathogenic viruses [5,6,7,8,9,10]. IFITM3 is involved in germ cell homing and maturation during embryonic development [11,12,13]. It has been reported that IFITM3 regulates viral infection through autophagy [14, 15]. However, its regulatory mechanism in retinal progenitor cells remains unclear and has not been reported.
Autophagy is involved in regulating the homeostasis of eukaryotic cells [16,17,18,19,20] and plays an important role in maintaining cell proliferation and self-renewal, especially in mesenchymal stem cells (MSCs) [21,22,23]. Here, we investigated the possible regulatory mechanisms of IFITM3 in mouse neural RPCs (mNRPCs) we established previously [3] through knockdown of IFITM3 expression. The data showed that knockdown of IFITM3 markedly inhibited mNRPC proliferation and cell viability through the chaperone-mediated autophagy (CMA) pathway. When IFITM3 was knocked down, cell proliferation decreased significantly, along with the disruption of the cell membrane transport system and destruction of the cell membrane structure, and the CMA pathway was continuously activated during this process. The results suggested that IFITM3 is a gatekeeper of progenitor cells that keeps the cells healthy during growth and self-renewal. In addition, RNA-seq data suggest that other regulatory factors, such as amino acid metabolism and fat metabolism, were also involved in regulating proliferation after IFITM3 knockdown, which suggests that inhibition of the expression of IFITM3 caused destruction of the cell membrane and eventually induced a metabolic crisis resulting in cell death. Taken together, these data indicate that IFITM3 is the first line of protection for RPC homeostasis and regulates mNRPC viability and proliferation mainly through the CMA pathway.
Materials and methods
Cell culture and RNA interference
Adult mouse neural RPCs (mNRPCs) established by our laboratory as our previous description [3] were plated onto dishes coated with 2% Matrigel (Corning, USA) and cultured in 1 × N2/B27 (Thermo Fisher Scientific, USA), 10 ng/ml bFGF (PeproTech, USA), 2 μm CHIR99021 (Selleck Chemicals, USA) and 0.11 mM 2-Mercaptoethanol (Thermo Fisher Scientific, USA) in DMEM/F12 medium (all other materials were from Thermo Fisher Scientific-Gibco, USA). To knock down IFITM3, commercially validated IFITM3 siRNA was synthesized by Proteintech (Rosemont, IL, USA) and transfected into cells using Lipofectamine 3000 (Thermo Fisher Scientific, USA) according to the manufacturer's protocol. After transfection for approximately 48 h, the cells were collected for further assays. The targeted sequences for the experiments are listed in Additional file 6: Table S1.
Cell viability assay
For the cell viability assay, cells were seeded at a density of 1 × 105 cells per 100 μL in each well in 96-well microtiter plates (Corning, USA) and transfected with siRNA for 48 h. Cells were grown in each medium in triplicate. Then, 10 μL of reagent from the Cell Counting Kit-8 (CCK-8, TargetMol, USA) was added to each well, and the plates were incubated for 3 h at 37 °C. Cell viability was measured as the absorbance at 450 nm with a microplate reader (iMark™ Microplate Absorbance Reader, BioRad, USA). The mean optical density (OD) values from triplicate wells containing each culture medium were used as the cell viability indices.
Real-time quantitative PCR
To obtain total RNA, cells were lysed with TRIzol reagent and treated with RNase-free DNase I (both from TaKaRa, China). Reverse transcription (RT) was performed according to our previously published article [3]. Then, quantitative RT-RCR was performed using the SYBR Green Master Mix system (Tiangen Biotech, China). Each PCR mixture contained 10 μL of 2 × SYBR Green Master Mix, sense and antisense primers (Sangon Biotech, China) at 5 μmol/μL, 10 ng of cDNA in a total volume of 20 µl and was subjected to the following protocol: 45 cycles of 95 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s. For relative quantification, 2−ΔΔCt values were calculated and used as an indication of relative expression levels. The IFITM3 gene primer sequences used for RT-PCR were 5′-TGTCCAAACCTTCTTCTCTCC-3′ and 5′-CGTCGCCAACCATCTTCC-3′. The internal control primer sequences for β-actin were 5′-GTGGACATCCGCAAAGAC-3′ and 5′-AAAGGGTGTAACGCAACTA-3′.
Immunofluorescence assay
Cells were fixed in 4% paraformaldehyde (PFA, Sangon Biotech, China) following the procedure described in our previously published article [3]. At the end of the experiment, the cells were stained with compatible Alexa 488- or Alexa 555-conjugated secondary antibodies (Thermo Fisher Scientific, USA) for 30 min at room temperature. The antibodies are listed in Additional file 6: Table S2.
Western blot analysis
Cells were harvested and homogenized in RIPA buffer (Beyotime, China) supplemented with protease and phosphatase inhibitor cocktails (TargetMol, USA) on ice and subjected to a previously described protocol [3]. The signal intensities of bands obtained were analyzed by ImageJ (NIH, USA). The antibodies used are listed in Additional file 6: Table S2.
Bioinformatics analysis
For the single-cell sequence analysis, all cells in the website (https://github.com/gofflab/developing_mouse_retina_scRNASeq) were clustered and visualized using the method of uniform manifold approximation and projection (UMAP) with Seurat (v4.0.4)∷RunUMAP [4, 24]. The annotations of all cells were provided in the dataset. The expression of IFITM3 gene in each sample were visualized using the function FeaturePlot in R package Seurat (v4.0.4).
For the RNA sequence analysis, RNA was extracted from mNRPCs and IFITM3-knockdown cells at 48 h with TRIzol reagent (TaKaRa, China). The RNA-seq library was sequenced with an Illumina NovaSeq 6000 PE150. The standard parameters |logFC|> 1 and P < 0.05 were used to screen differentially expressed genes (DEGs). The R package clusterProfiler was used for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. The results were visualized with GOplot.
Organelles involved in IFITM3-knockdown cells
After 48 h of transfection with IFITM3 siRNA, cells were treated with or without 100-nM rapamycin (RAMP, Selleck, USA) for 4 h then analyzed after incubation with 50-nM LysoTracker (Beyotime, China), 50-nM MitoTracker (Beyotime) and 1-μM ER-Tracker (Beyotime) for 30 min. The nuclei were stained with Hoechst 33342 (Sigma-Aldrich, Germany).
LC3-GFP-mCherry construction and infection and autophagy flux assay
The Ad-mCherry-GFP-LC3 plasmid was purchased from Addgene (plasmid #110060), amplified and sequenced following instructions from Addgene using the following sequencing primers: forward: 5′-CGCGGATCCGGTCGCCACCATGGTGAGCAAGGGCGAG-3′, reverse: 5′-CGCGGCGCGCCGCTGGGTCTAGATGCATGC-3′. The PCR fragment was sequenced to verify that no errors had been introduced. LC3-GFP-mCherry lentivirus was produced by transfection in 293 T cells. The 293 T cells were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS, ExCell Bio, China). The virus-containing supernatant was collected after 48 h of transfection and subjected to a previously described protocol [3]. GFP and mCherry expression was verified by microscopy. The cells were treated with 100-nM RAMP to study autophagic flux after IFITM3 knockdown for 48 h. Then, the expression of GFP and mCherry was observed after staining with Hoechst 33342 (Sigma-Aldrich, Germany).
Statistical analysis
All data are expressed as the mean ± SEM. All analyses were performed with GraphPad Prism 9.3 software. One-way ANOVA was employed for the statistical comparisons. A value of P < 0.05 was considered to indicate statistical significance.
IFITM3 is involved in mNRPC proliferation
Initially, to obtain information on the variation and distribution of the IFITM3 gene during the development of the mouse retina in vivo, we integrated a published dataset of the developing murine retina in single-cell RNA sequencing and visualized the cell-type identification in Fig. 1A [4]. During the embryonic stage, the IFITM3 gene was mainly expressed in the early RPCs at embryological days 11 (E11) and E12 (Fig. 1B). For the postnatal stage (P0, P2, P8), fewer positive expressed in the detected cells, but it also expressed in RPCs and Müller cells 14 days after birth. These results suggest that IFITM3 is important for retinal progenitor cells in retinal development.
To investigate the function of IFITM3 in mNRPCs, we transfected siRNA IFITM3 into the cells and examined cell viability. Cell growth (Fig. 1C–E; Additional file 1: Figure S1A-S1C) was clearly inhibited in the IFITM3 siRNA-transfected group (siRNA IFITM3) compared with the c group and scramble control (SC) group, with significantly decreased expression of IFITM3 at the RNA (Fig. 1F) and protein levels (Fig. 1G) after IFITM3 knockdown, but there was no significant decrease in ARPE19 cell growth (Additional file 1: Figure S1D-E). The cell viability was greatly decreased after IFITM3 knockdown, as shown by the CCK-8 assay, which significantly differed among the groups (Fig. 1H), and the protein expression of Cyclin D1 (CCND1) decreased in the corresponding cell groups (Fig. 1I). These results indicated that IFITM3 was not only involved in the regulation of cell division and activity of retinal progenitor cells during retinal development but also involved in the proliferation and division of in vitro cultured RPCs.
Cell membrane structure and function were dramatically changed after IFITM3 knockdown
To further explore the regulatory effect of IFITM3 knockdown on the cells, RNA-seq analysis was performed. Among the GO terms, cell membrane and extracellular matrix (ECM)-related, transmembrane transport-related and synaptic transmission enrichment-related terms were the most enriched after knockdown of the IFITM3 gene. These results revealed that genes related to ion transmembrane transport, vacuoles, lysosomes and cellular calcium ion homeostasis were upregulated in the knockdown group, while genes involved in regulation of the ECM, WNT pathway and cell proliferation were significantly downregulated (Fig. 2A), suggesting that the stability of the cell membrane system was seriously damaged and that its material transport function was blocked, ultimately leading to cell death because of constant nutrient deficiency. These findings further reveal how IFITM3 is involved in regulating the proliferation of mNRPCs. The KEGG pathway enrichment assay further revealed that the signaling pathways enriched in the DEGs mainly included the synaptic vesicle cycle, lysosome pathway, calcium signaling pathway, MAPK signaling pathway and other upregulated pathways (Fig. 2B; Additional file 2: Figure S2A), as well as downregulated pathways, such as the WNT, amino acid biosynthesis and fatty acid metabolism pathways (Fig. 2C; Additional file 2: Figure S2B).
Furthermore, GO analysis of the ECM pathway showed that the expression of ion channel-related genes, including the genes encoding calcium (such as CACNB2), potassium (such as KCNB1, KCNF1 and KCNC3) and chloride (such as CLCN1) ion channels, was clearly changed after IFITM3 knockdown (Additional file 2: Figure S2C); additionally, genes that regulate cell proliferation involved in metabolism (such as TNC and MVD) and proliferation-related pathway genes (such as CCND3, STAT1 and IL6) (Additional file 2: Figure S2D) also showed significant changes in expression, indicating that inhibition of IFITM3 expression led to damage to the cell membrane, abnormal material transport, abnormal metabolism of intracellular substances, decreased cell proliferation and ultimately cell death. Taken together, these results indicate that the integrity of the membrane system (including the cell membrane, vacuole, lysosome and ECM) was impaired after IFITM3 knockdown and that cells could no longer survive without sufficient nutrition, eventually leading to cell death.
Lysosome activation in IFITM3-knockdown cells
Because membrane-related systems were severely damaged after IFITM3 knockdown, we investigated changes in membranous organelles, including the endoplasmic reticulum (ER), mitochondria and lysosomes in the cells. The lysosome is an important site of regulation for mTORC1 signaling, which mainly controls eukaryotic cell growth. We treated cells with 100 nM RAMP, a specific mTOR inhibitor and an autophagy activator, and then treated the cells with 50-nM LysoTracker, 50-nM MitoTracker and 1-μM ER-Tracker for 30 min. Lysosomes showed agglomeration in IFITM3-knockdown cells without RAMP treatment, while lysosomes showed agglomeration in all RAMP treatment groups, but there was no significant difference in IFITM3-knockdown cells with or without RAMP treatment (Fig. 3A). Mitochondria and ERs were increased in IFITM3-knockdown cells treated with RAMP, but there was no significant difference among the groups (Fig. 3B, C), suggesting that mitochondria and ERs were not involved in regulating mNRPC proliferation after IFITM3 knockdown and microautophagy (mA) was not activated.
The lysosome-specific markers lysosome-associated membrane protein 1 (LAMP1) and LAMP2A were detected in RAMP-treated and untreated cells, which showed that the expression of LAMP1 (Fig. 4A) and LAMP2A (Fig. 4B) was significantly increased and accompanied by significantly activated lysosomes in RAMP-treated cells, especially in IFITM3-knockdown cells (Fig. 4A, B), suggesting that the lysosomal system is activated immediately following the destruction of the cell membrane system, eventually causing cell death. These results suggest that IFITM3 in the cell membrane provides the first protective barrier for mNRPCs, and its decreased expression leads to the breakdown of cell homeostasis and initiation of lysosome formation to eliminate damaged cells. Furthermore, there was no significant difference among the IFITM3-knockdown cells with or without RAMP treatment, suggesting that MA was not activated in the cells when the IFITM3 gene was knocked down. However, IFITM3 knockdown led to a cascade of effects causing increased membrane permeability, activation of lysosomes and the CMA pathway, eventually leading to cell death.
Activation of the CMA pathway when IFITM3 is knocked down
Cell viability was significantly inhibited and eventually caused cell death after IFITM3 knockdown. Therefore, signal pathways regulating cell proliferation including mTOR pathway and MAPK pathway were detected, the data showed no significant activation of the mTOR pathway (Fig. 5A), but obviously decreased expression of ERK1/2 among the groups (Fig. 5B), suggesting that the downregulation of IFITM3 resulted in decreased cell survival and that the ability of the cells to proliferate was severely reduced.
Then, the results of western blot analysis showed significantly increased expression of the antiapoptotic protein BCL2 (Fig. 5C, F) and decreased expression of P53 (Fig. 5C) in IFITM3-knockdown cells, which suggested that the apoptotic pathway was not activated when IFITM3 was knocked down. Additionally, we examined apoptotic cells by staining the cells with Annexin V-PE, the results showed no obvious apoptosis in IFITM3-knockdown cells (Additional file 3: Fig. S3A–C), and no apoptotic bodies were detected by Hoechst 33342 staining (Additional file 3: Fig. S3D–F), which consistent with WB results.
Next, the further investigation focused on the MA and CMA pathways. The expression of MA pathway-related proteins is complicated, there was no significant change in ATG13, BECLIN1, ATG5, ATG7 and LC3A/B, but P62 and LAMP1 were significantly upregulated (Fig. 5D, F), the increased LAMP1 suggested the formation of autophagy lysosomes, and the results suggested that the MA pathway was not activated after IFITM3 knockdown. Therefore, MA is not the pathway of this regulatory effect.
However, the expression of LAMP2A and HSC70 (Fig. 5E, F) was significantly upregulated, which indicated and confirmed the activation of CMA in the cells after IFITM3 knockdown. Although the mRNA expression (Additional file 4: Fig. S4) was not consistent with the protein expression after knockdown IFITM3 for 48 h, the specific reasons need to be further studied in the future. To further confirm the activation of MA or CMA, the expression of LC3A/B in IFITM3-knockdown cells was analyzed with immunohistochemical (IHC) first, the results showed that there was no significant difference in the cells (Fig. 5G). Then, the generation of autophagic flux was examined by constructing stable LC3-GFP-mCherry-expressing mNRPCs. The results showed that no autophagic flux was generated in the cells (Fig. 5G; Additional file 5: Fig. S5).
Some of the above results were consistent with IHC results. When IFITM3 was knocked down (Fig. 6A–C), the results of IHC analysis showed slightly increased expression of BCL2 (Fig. 6D–F) and no significant changes in ATG7 expression (Fig. 6G–I), while the expression of LAMP1 (Fig. 6J–L), P62 (Fig. 6M–O) and LAMP2A (Fig. 6P–R) was significantly changed. The data were consistent with the WB results and IHC data during lysosome activation, indicating no activation of MA or the apoptotic pathway, while the CMA pathway was activated in IFITM3-knockdown cells. According to the above data, we believe that IFITM3 regulates cell viability mainly by regulating the CMA pathway, suggesting that the IFITM3 gene has a protective function that increases progenitor cell survival and self-renewal.
Stem cell-related cell therapies have developed rapidly around the world. In the field of cellular ophthalmology therapy, safety data have emerged from several phase I/II clinical trials of cell transplantation with retinal pigment epithelial (RPE) cells or MSCs [25,26,27,28,29,30,31,32,33,34,35]; however, some issues, such as host-graft rejection, validation over too short a duration (less than 12 months) and a lack of clinical significance, remain. Although RPC transplantation is considered a very promising approach [2, 36,37,38], its application is limited because of insufficient donor cells. Therefore, the need to obtain enough seed cells has become urgent. Previously, we established mNRPCs that can be cultured long-term in vitro and maintain their self-renewal properties based on the combination of bFGF and CHIR99021 [3]. In further biological studies, RNA-seq data showed the high expression of some genes that are expressed at low levels in adult retinal tissue, such as IFITM3, an intrinsic host factor with extensive function in the organism, including antiviral function [39, 40], immune function [41,42,43,44] and germ cell specification [45] that is localized in several cellular components, including the apical part of the cell, cell surface and endolysosomal membrane. Currently, limited reports on the role of IFITM3 in stem cells are available, and its specific function in the field is still unclear. Here, we studied the function of the IFITM3 gene in mNRPCs by knocking it down, and we focused on investigating the changes in the autophagy pathway, which is very important to cell death and homeostasis. In mNRPCs, we found that chaperone-mediated autophagy (CMA) plays a very important function in regulating retinal progenitor cell homeostasis.
Our analysis provides a series of studies of signaling pathways involved in regulating cell growth and death, and mNRPCs showed significantly decreased cell viability and proliferation after IFITM3 knockdown. The significantly decreased expression of CCND1, a key molecule in the regulation of cell cycle progression from G1 to S phase, suggests that IFITM3 affects the proliferation of mNRPCs by regulating cell division. In addition, the expression of ERK1/2 was decreased accordingly after IFITM3 knockdown, indicating that IFITM3 regulates cell proliferation and may be associated with the MAPK pathway. And the apoptotic pathway was not activated when IFITM3 was knocked down. Further detection revealed that the CMA pathway was activated with high expression of HSC70 and LAMP2A, although the mRNA expression was not consistent with the protein expression. It has been reported that CMA began to increase at 8 h after serum starvation and peaked at 36 h, lasting for 3 days [46]. Overall, our work provides a basis for understanding how IFITM3 regulates RPC proliferation and homeostasis.
As one of the important members of the autophagy family, macroautophagy (MA), mitophagy (mA) [47,48,49] and CMA [49,50,51,52], CMA starts with HSC70, and HSC70 targets cargo proteins and then binds to LAMP2A to translocate the proteins into the lysosomal lumen. CMA activity is detectable under basal conditions in most cells, but overactivation occurs during stress, such as nutrient deprivation [53] and lipid overload [54, 55]. Consistently, our IFITM3 knockdown cell model demonstrated that IFITM3 maintains RPC cell homeostasis by stabilizing the cell membrane or cellular membranous organelles. Consistent with the LAMP2A immunolabeling data, WB and lysosome activation assays confirmed the overactivation of CMA. Moreover, RNA-seq data showed significant changes in the membrane system, and GO and KEGG analysis of the RNA-seq results suggested that IFITM3 is involved in regulating mNRPC proliferation by changing the structure and function of the cell membrane. Additionally, lysosomes also showed a marked increase in abundance in this abnormal membrane system. Our results suggest that CMA activation is due to the instability of the cell membrane system, lysosomal activation and accumulation caused by IFIMT3 knockdown.
Knowing that lysosomes are a key factor in autophagy and important for regulating mTORC1 signaling [56,57,58,59], the main regulator of autophagy, especially MA, we studied changes in lysosomes and the autophagy pathway after RAMP treatment when IFITM3 was knocked down. An increase in the expression of LAMP1 and LAMP2A in knockdown cells suggests the activation of lysosomes, consistent with the LysoTracker results. We believe that IFITM3 regulates cell proliferation mainly by activating the CMA pathway, and lysosomes are the key member of this regulatory process. Some similarity exists between our data and another study that observed protein quality control in quiescent steady-state hematopoietic stem cells (HSCs) of CMA [52].
Taken together, this evidence suggests a protective role for IFITM3 interactions in mediating self-renewal and cellular homeostasis in mNRPCs. The decreased expression of IFITM3 in mNRPCs resulted in the destruction of the cell membrane system and activation of lysosomes, a key factor in the autophagy pathway. Similar to our findings, IFITM3 has been reported to shuttle virus particles directly to lysosomes [60]. In mNRPCs, lysosomes were activated to remove the stress damage in cells caused by IFITM3 knockout and accompanied by activation of the CMA pathway, and continuous overactivation of CMA caused cell death.
Overall, the study provides novel insights into the role of IFITM3 in RPCs, such as solving the problem of maintaining self-renewal in vitro by regulating the CMA pathway or attempting to activate quiescent in vivo RPC cells to repair damaged retinas in retinal degenerative diseases.
In this report, we found that IFITM3 plays an important role in RPC proliferation by activating the CMA pathway, suggesting that the low-level activation of CMA is important in maintaining the homeostasis of RPCs, while the overactivation of CMA leads to cell death.
Availability of data and materials
RNA-seq data generated in the study can be accessed at the Gene Expression Omnibus under accession code GSE176472(https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE176472). The original gel images of the western blot were supplied in the supplementary material (supplementary material Original gel images). All other data are included within the article and its additional files.
Blank control
Cyclin D1
Chaperone-mediated autophagy
Differentially expressed gene
Extracellular matrix
Endoplasmic reticulum
Gene ontology
Interferon-induced transmembrane protein 3
Kyoto encyclopedia of genes and genomes
Lysosome-associated membrane protein 1
- mA:
- mNRPC:
Mouse neural retinal progenitor cell
Mesenchymal stem cells
Optical density
Retinal progenitor cell
Scramble control
Uniform manifold approximation and projection
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This paper was supported by the National Natural Science Foundation of China (32070719, 81770942), and China Postdoctoral Science Foundation (2019M661631).
Author information
Authors and Affiliations
CJ and LL conceived and designed the experiments, performed the experiments, analyzed the data, and wrote the paper. QO and ZW collected and analyzed the RNA-Seq data. CJ, QO, HT and G-T X contributed to design the experiment, financial support, manuscript writing and final approval of manuscript. TW and JC performed the experiments. JZ, YW, J-Y X, FG, JW and JL contributed to reagents/materials/analysis tools. All authors have read and approved the manuscript.
Corresponding authors
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Ethics approval and consent to participate
This study is a continuous work of our previous study (https://doi.org/10.1186/s13287-018-1091-y). All the retinal progenitor cells of this study are maintained from our previous study (https://doi.org/10.1186/s13287-018-1091-y). There were no other animal involved in this manuscript.
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The authors declare no competing interests.
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Supplementary Information
Additional file 1.
Figure S1: Cell morphology and expression of IFITM3 of mNRPCs after knockdown with siRNA for 48 h.
Additional file 2.
Figure S2: High-throughput sequencing analysis of IFITM3-knockdown cells.
Additional file 3.
Figure S3: Cell apoptosis assay after IFITM3-knockdown for 48 h.
Additional file 4.
Figure S4: qRT–PCR assay of LAMP2A and HSC70 expression in mNRPCs after knockdown IFITM3 for 48 h.
Additional file 5.
Figure S5: Fluorescent images showing mCherry-GFP-LC3 expression after IFITM3 knockdown used to assay autophagic flux in the cells.
Additional file 6.
Supplementary Material.
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Jin, C., Ou, Q., Chen, J. et al. Chaperone-mediated autophagy plays an important role in regulating retinal progenitor cell homeostasis. Stem Cell Res Ther 13, 136 (2022). https://doi.org/10.1186/s13287-022-02809-z
DOI: https://doi.org/10.1186/s13287-022-02809-z


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Answer: b. Non-directive
Psychotherapy is simply the process of treating individuals who have been diagnosed with mental and emotional dysfunctions, where different kinds of psychological techniques are applied during treatment.
"Non-directive" is the word that best describes a therapist's role in psychotherapy when humanistic values are considered. This manner of providing effective solutions in psychotherapy aims to enable the client to create a stronger and more powerful sense of self-belief, self-love, and self-support.
This approach helps the client to have a more positive view of life and to live a happier lifestyle.

subset: commoncrawl


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First Trimester
Pregnancy is full of anatomical, physiological & biochemical changes to support the growth & development of your fetus. These changes begin soon after fertilization and continue throughout your pregnancy.
The 1st trimester is a period of ups and downs. Some days may feel great, and others miserable. Every woman's experience of each pregnancy is unique.
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Restful sleep happens with a rested mind. The Huffington Post recently suggested five ways in which we can power down when it's time for rest.
You know you should sleep more (which could happen), walk more (which should happen), and turn off your phone (that's not happening). Here are a few surprising ways to quiet your brain at different times of day — without going off the grid.
Click here to view original web page at www.huffingtonpost.com


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These are the steps you need to take in order to set up a Returned Check. You can also learn about how part-time employees and contractors report their income with QuickBooks online.
In QuickBooks, you can record a returned vendor payment as a credit. The process is similar to recording a return of your own check.
In QuickBooks Online, you must record a drop to the bank account for the whole amount of the check in order to correctly report a bounced check. The bank will then charge you a non-sufficient funds (NSF) fee, which you must record. Finally, you must charge the consumer for the returned check cost.
In QuickBooks online, how do I record a returned customer check?
What you'll need to accomplish is:
- Use Write Check to enter the returned check.
- Make a payment input change.
- Create service items for fines and rejected checks.
- Make an invoice for the charge for a bounced check.
- Fill in the bank service fee.
- Print the statement and mail it to your client.
- Keep track of your customer's new payment.
Also, in QuickBooks, how do I report a bounced check that I wrote? Take care of a bouncing check you wrote.
- Select Make General Journal Entries from the Company menu.
- Make sure the Entry No. and Date are right.
- Charge the amount of the NSF check to the checking account.
- The amount of the NSF check should be credited to the A/P account.
- Fill in the Supplier's name.
- Enter memos and classes if desired.
- Select Save & Close.
How do you record a returned check in accounting, for example?
The returned check is not shown in the depositor's accounting records.) This implies that the depositor must 1) credit Cash and 2) debit the account that was credited when the check was first received. Frequently, the depositor's bank will impose a fee for processing the returned item.
In QuickBooks, how do I classify a returned check?
- Choose Expense.
- Payee? from the drop-down menu:
- Select the account you use to pay expenditures from the Payment account? drop-down box.
- (Optional) In the Ref no. field, type "NSF fee" to separate it from other charges.
- Select Bank Charges from the Category column.
- Fill in the total amount you were charged.
Answers to Related Questions
In QuickBooks, how do I report inadequate funds?
Option 1: Use the Bounced Check Recording function.
- Select Customer Center from the Customers menu.
- Choose the Received Payments option from the Transactions tab.
- Double-click the payment you wish to mark as non-sufficient funds.
- Select the Record Bounced Check icon on the Main ribbon tab in the Receive Payments window.
In QuickBooks Online, how do I record an NSF check?
To record a bounced check in QuickBooks Online, follow the five steps below:
- Make a note of the decrease in the bank account balance.
- Select the Expense Menu from the drop-down menu.
- In QuickBooks Online, enter the NSF Fee Expense.
- In QuickBooks Online, go to Invoices.
- The NSF Fee will be billed to the customer (Optional)
What happens if a cheque is returned non-sufficient funds?
The check may bounce if the issuer does not have enough money in his or her account to cover it by the time it clears, in which case it will be returned to the payee who attempted to cash it. It costs money to write or receive a bounced check, often known as a nonsufficient funds, or NSF, check.
How can I make an entry in the NSF journal?
The first step is to make a diary note for the bounced check:
- Choose the + New option.
- Select Journal Entry from the drop-down menu under Other.
- In the Journal date section, write the date the check bounced.
- Select Receivables (accounts receivable) from the Account option on the first line.
- Enter the amount of the bounced check in the Debits column.
What happens if a cheque is returned because there aren't enough funds?
If you don't pay the amount of a bounced check within the time range specified by your bank, your account may be closed. The check bounces and is returned to the depositor's bank if your financial institution fails to cover it. A nonsufficient funds fee, commonly known as an NSF or returned item fee, will almost certainly be applied to your account.
In QuickBooks online, how do I record a returned vendor payment?
Create a bill credit for the products that were returned:
- Select Enter Bills from the Vendors menu.
- To account for the return of items, choose the Credit radio choice.
- Fill in the Vendor's name.
- The Items Tab should be selected.
- Enter the same amounts for the returned products as the refund check.
- Select Save & Close.
Why is the checkbox for record bounced greyed out?
Change the payment mode to check whether the Quickbooks record bounced check button is greyed out. You have an ACH item, which is why the bounced check button is dormant. These things are usually paid for with cash or a credit card. Changing the payment mode to record a returned check is as simple as that.
What are the signs that my check has bounced?
Check the amount of your check versus the funds in your checking account. Subtract the amount of the check from your checking account's available balance. Your check may bounce if the value you get is negative or you find that you have less money in your account than the check was issued for.
What is the entry for a bounced check in the journal?
To enter via journal entry, follow these steps:
Select Journal Entry from the "+" menu. Fill in the date on which the cheque bounced. Select Receivables (accounts receivable) from the Account column. Enter the amount of the bounced check under Debit.
What is the Entry in the NSF Check Journal?
Entry in the NSF Check Journal
Account | Debit | Credit |
Receivables (accounts receivable) | 250 | |
Cash | 250 | |
Total | 250 | 250 |
What is the journal entry for an NSF check adjustment?
Adding and Removing Entries
Post your interest credit received from the bank as a debit to your cash account and a credit to your interest revenue ledger account. Post returned check fees, NSF fees, to your Receivables (accounts receivable) account along with the amount of the check that was returned.
In which journal do you record a cheque that has been returned unpaid?
If you make a payment and the check is returned, the following diary entry will be written in the book: 1 if the payment is paid by check
In QuickBooks, where is the button for bounced checks?
Select "Customers| Receive Payments…" from the Menu Bar to record bounced checks in QuickBooks Desktop Pro. Then locate or browse to the individual customer payment that includes the bounced check that you received. Then, on the "Main" tab of the Ribbon at the top of the window, click the "Record Bounced Check" button.
Is a bounced check the same as a returned ACH?
Non-sufficient funds ACH messages are treated the same as checks. When the bank sends you a notification of NSF/Closed Account/Stop Payment, the cheque is declared non-sufficient funds/returned. This normally happens within two to three days after the deposit being made.
In QuickBooks, how do I report a bounced check to a vendor?
Take care of a bouncing check you wrote.
- Select Make General Journal Entries from the Company menu.
- Make sure the Entry No. and Date are right.
- Charge the amount of the NSF check to the checking account.
- The amount of the NSF check should be credited to the A/P account.
- Fill in the Supplier's name.
- Enter memos and classes if desired.
- Select Save & Close.
What is the location of the create icon in QuickBooks?
Start QuickBooks and go to your business file. To access the Customize Icon Bar window, right-click the icon bar and pick "Customize Icon Bar" from the menu. To access the Add Icon Bar Item window, click the "Add" button.
In QuickBooks, how can I correct a returned check?
Option 1: Use the Bounced Check Recording function.
- Select Customer Center from the Customers menu.
- Choose the Received Payments option from the Transactions tab.
- Double-click the payment you wish to mark as non-sufficient funds.
- Select the Record Bounced Check icon on the Main ribbon tab in the Receive Payments window.


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Can You Study Interior Design In College. If you're interested in studying a interior design degree in canada you can view all 39 bachelors programmes. From color schemes to floor plans to building codes, interior designers do it all.
The tuition fee amounts to 26, 345 eur per year. In 2020, 26 interior design students graduated with. You will get to study from boston's top artists and.
The Benefits Of Studying Architecture Or Interior Design.
Bachelor of arts in interior designing is a comprehensive course of three year duration and was meticulously designed for in depth studies in various fields of interior. They give lectures, assign papers and. From color schemes to floor plans to building codes, interior designers do it all.
The Ba (Hons) Interior Design Programme, In Particular, Only Has Around 30 Students A Year, Giving Professors The Chance To Fully Support Their Individual Abilities.
International applicants need to pay an amount of £26,000 per year for 3 years. You can also read more about interior design degrees in general, or. The oxford school of architecture offers a 3 year undergraduate degree in interior design.
Arrange Your Free Consultation Online Or At Our London Office Today.
You can also read more about interior design degrees in general, or about. What colleges offer interior design classes in 2022? Interior designers transform dull impractical rooms into lively, functional spaces for various purposes.
Phone +27 (0)12 420 4542.
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If The Student Is Opting For The Placement Year, An Additional Amount That Is 20% Of The Total Fees Has.
There are 272 colleges in the u.s. One good reason to study here is because of its location. It was founded in 1916.


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The City of Stockholm and IBM are working together in the GrowSmarter Project in the large event area in Stockholm to use new technology to provide a better event experience for the visitors and support decisions to handle incidents, improve mobility and reduce emissions from traffic.
- Capture new real-time sensor data on the flow of people and vehicles at large events into the Watson IoT platform
- Use advanced analytics, visualization and AI to deliver real-time transport advice to the visitors and vehicle drivers.
- Provide benefits to the event organizers, the traffic operations center and the city transport planners from new insights for decisions and impact follow up.


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Mr. G. Lafferty - English Master, 1961-75
Sadly, Gerry Lafferty died on August 15th, 2005, aged 75. Tributes and memories will be posted here as they come in. Also, check the Guestbook for more memories.
Another Photograph at the December 2001 reunion
Tributes in chronological order of years at HCS:
From Nigel Morley
Dear Mrs Lafferty,
May I pass my condolences to you and your family on the death of Gerry.
You do not know me and indeed, I never met or spoke to your husband after leaving school. When Gerry arrived at Harrow, he was like a breath of fresh air compared to the reactionary and Edwardian attitudes at the school. Although I took sciences, he was an inspiration to myself and my peer group.
He and a fellow colleague treated the boys as adults, rather than cannon fodder for the school statistics on University and Oxbridge entrance. Gerry was one of the few people to write anything complimentary on my school report. He kindly noted that although school played very little part in my life, society would hear more of me in the future.
I hope he put the same words on my best school friends report, Paul. Sir Paul Nurse as he is now, is President of the Rockefeller Institute in Manhattan and a Nobel Laureate of Medicine. Such was the nature of the boys Gerry taught and even some 40 years later, we still have fond memories of his inspirational nature, which was sometimes at odds with the draconian environment we both found ourselves in.
Over the years the Gaytonian website has constantly lauded your husbands achievements. However, your husbands greatest achievement is in the success of hundreds of pupils he taught, but perhaps more importantly, their fond memories and respect for a decent man.
Finally, once again my sincerest condolences to you and your family.
Yours sincerely,
Nigel Morley, MRPharmS
From Richard Salter, QC
What do I remember of Gerry Lafferty? A flurry of gown, chalk and inhaler – the Guinness logo on the key ring - a musical, emphatic, Scots burr of a voice – lists of new authors on the blackboard – reading Henry V in class – Julius Caesar on stage - the passion when talking of the things he cared about.
So many of us who passed through HCS in the 1960s owe so much to Gerry's wisdom and humanity: his enthusiasm for life, as well as for literature; his ability to instil self-belief in the boys he taught.
Gerry taught us well: but, most of all, he taught us to think for ourselves. He exhorted us to read the Times (in all its pre-Murdoch establishment pomp), but also to subscribe to the New Statesman (then at its subversive height). He set us challenges. Read (and be able to talk intelligently about) Portrait of the Artist, and I will trust you with my copy of Ulysses (this was before Penguin brought out the paperback). Concentrate on Pattern of Islands and Silas Marner in term time, and your holiday reading can be Fahrenheit 451, Catcher in the Rye, and Player Piano. Memorise the characteristics of Milton's minor verse, and I will show you lyrics by Donne and Shakespeare sonnets that will catch at your heart.
Gerry showed us that books could be doors into other people's worlds, and that even the critic's most dryly technical tools could be the keys to unlock them. Books were fun. Books were there to challenge us, and to be challenged. Nothing was too sacred to be questioned.
Gerry was a great teacher. He kindled in me a joy in literature that propelled me through Oxford, and that stays with me still. I count myself truly fortunate to have had the privilege of being taught by him.
Richard Salter
(HCS 1963-70)From Stephen Frost:
Gerry Lafferty was probably the best English master who ever taught me. A great man, approachable and kind.
We got through so many set books in the early 1960s, many of them classics that have probably disappeared from today's English syllabus: The Oak Settle, The 39 Steps, Kidnapped, The Last of the Mohicans, Prester John, The Cruel Sea, A Kid for Two Farthings, David Copperfield, Silas Marner, Great Expectations, A Pattern of Islands and Diary of a Nobody.
And an awful lot of Shakespeare: Julius Caesar, The Merchant of Venice, Macbeth, Henry IV Parts I and II, Henry V and Hamlet. "Julius Caesar is like an onion, the more you peel off, the more you find …"(first form). "Macbeth is like an onion, the more you peel off, the more you find…"(third form). "Hamlet is like an onion, the more you peel off, the more you find…" (sixth form). And so much poetry, Masefield, Thomas, Pope.
During the very hot summer of 1967, I remember him teaching us the theory of English poetry in B4 before it was converted into the language lab. Trochees, spondees, dactyls and iambs. Blank verse and heroic couplet.
Gerry drove in from Gerrards' Cross every day in his grey VW. He was often late in the morning and missed assembly. Was that due to the distance involved or Gerry's small protest at something he disliked? In the sixth form 1968-70, he arranged for budding lefties and dissidents to be able to subscribe to New Statesman, surely something that would not have been permitted during Square's regime. Gerry was something of a non-conformist, and that was one of the reasons many of us liked him.
So farewell then Gerry Lafferty
You were the greatest English (er, Scottish) master who taught me
How to read, think and spell
Though now I ma leraning how to use a smell checker
(apologies to E.J. Thribb)
Gerry told us he never put pupils in detention and he never used corporal punishment. How many other masters would say that to their charges? Another measure of his qualities.
He was a man take him for all in all.
We shall not look upon his like again.
Stephen Frost (1963-70)
From Katie Finch (Percival)
Very sad news: the teachers who really mattered in my education were not those at the Girls School, who mostly bored and exasperated me, but those I met through convergence and the Old Gayts Drama Club at the Boys School. Jim Golland, Fred Bilson, Gerry Lafferty and the mighty Harry Mees. It was a delight to be involved with Gerry's production of Hamlet: always supportive, encouraging, entertaining and boy,could we pack a lot of costumes into his volkswagon beetle. I shall long treasure the memory of him at the Drama Reunion: we had one chance for a read through of the script before the event, so compere Francis Matthews and I went into the Art Room (formerly the backstage cloakroom/S1/S2) to run through it and Gerry came along too. Perched on a stool, laughing at all the terrible jokes, offering advice and having a good time. He made it such fun- but then, he always did.
From Jon Grunewald:
Actually I attribute most of what I have achieved in life to Gerry Lafferty, if only because I had little academic ability other than in English, and I don't think I'd have made it to Oxford to study English Literature without his encouragement (given liberally to all his pupils) and his skill in teaching us. He was one of those "captain, my captain" teachers. And better than Robin Williams. One of his gifts was to teach us to recognise bad poetry for what it was. That was quite a shock at first, because schoolboys are usually encouraged to treat all poetry with reverence and fear. If you could manage to understand the author's intention, you could then admire the poem. Never ridicule it. But Jock Lafferty read us the poetry of William Macgonagall ("alas noble Prince Leopold, he is dead! Who often hath his lustre shed". Shed?!) and I have a recollection of one wonderful lesson when he took us through a poem which began with something about the jonquils blooming in Samarkhand and finished "and Stratford's church guards dearer dust, Than Omar's shrine". We weren't sure what to make of it at first. It was a poem that rejoiced in the fact that our nation had given birth to Shakespeare. Yes, said Jock, in a comical parody of a Scottish housewife, it's simply telling us "WE'VE got the BEST". And we could then see through the images, the assonance and the iambic rhythmns, to the shallow sentiments inside the poem. It was probably afterwards that I got into trouble with Jago for making fun of some lines in Wordsworth... still, Jock's encouragement to us to question authority, read a good newspaper and to enjoy art rather than fear it, was something we all took away with us into our future lives. As Jock would have said: am I right or am I wrong? It was nice to be asked - not every teacher did. And he was usually right, anyway.
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Use the influxctl CLI to query data
Limited availability
InfluxDB Clustered is currently only available to a limited group of InfluxData customers. If interested in being part of the limited access group, please contact the InfluxData Sales team.
Use the influxctl query
to query data in InfluxDB Clustered with SQL.
The influxctl query
command only supports SQL queries; not InfluxQL.
Provide the following with your command:
Database token: Database token with read permissions on the queried database. Uses the
setting from theinfluxctl
connection profile or the--token
command flag. -
Database name: Name of the database to query. Uses the
setting from theinfluxctl
connection profile or the--database
command flag. -
SQL query: SQL query to execute. Pass the query in one of the following ways:
- a string on the command line
- a path to a file that contains the query
- a single dash (
) to read the query from stdin
influxctl query \
--token DATABASE_TOKEN \
--database DATABASE_NAME \
"SELECT * FROM home"
influxctl query \
--token DATABASE_TOKEN \
--database DATABASE_NAME \
cat ./query.sql | influxctl query \
--token DATABASE_TOKEN \
--database DATABASE_NAME \
Replace the following:
: Database token with read access to the queried databaseDATABASE_NAME
: Name of the database to query
Output format
The influxctl query
command supports the following output formats:
Use the --format
flag to specify the output format:
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Almost everyone in the country has read or watched something written by Ismat Chughtai. She wrote Garam Hawa (with Kaifi Azmi), which is considered to be a cinematic classic, but she most renowned for her short-story Lihaaf, which had homosexual themes, a very bold thing back in her day.
We recently stumbled upon an old interview of hers with Prasar Bharti, and its all kinds of warm. In the interview, she talks about her parents, and how her father loved her mother so much, he used to say that he could easily live his life with her without the kids.
Hamare abba kehte the ki tum sab na hote, tab bhi hum apni begum ke sahaare ji sakte the.
She also discussed how she got a secular upbringing, which was rooted in reality. For instance, in a hilarious bit, she tells that her parents used to talk if they should celebrate Diwali, and used to go ahead with it, hoping it will bring prosperity.
Abba ne amma se kaha, "Diwali kaahe ke liye?". (Toh amma boli) "Laxmi aati hai". Toh unhone kaha, "Laxmi tumhare yahan kyun ayengi, tum musalman ho", aur amma ne kaha, "Arey unn bechari ko kya maloom kaun Hindu hai, kaun musalman, woh jahan roshni dekhengi chali aayengi".
She further talked about Shaheed Lateef, her husband from 1942–1967, and how he would say that she shouldn't handle money matters but she did anyway. She also pointed out that she was better at saving money than him.
When asked about her writing influences, she said that she admired a lot of people, but never copied them. Manto was one of them, and the other being George Bernard Shaw.
Sab kehte the Bernard Shaw ko maine ghola aur pi gayi.
But she added what she understood about writing a story – There has to be a beginning, a middle, and an end.
You know? Simple. Like herself, and this beautiful interview. You can watch it here:


text:
Yuma, Arizona - This morning, at approximately 9:37 a.m., Yuma Police officers responded to a report of an armed robbery at the Washington Federal Bank located at 100 E 32nd Street.
The initial investigation revealed an unknown male subject entered the bank and approached the counter. The suspect displayed a handgun and demanded money and car keys. The suspect took an undisclosed amount of money, the employee's car keys, and fled the scene in the employees' vehicle, a white Nissan Sentra. The vehicle was located by officers, unoccupied, about a block from the scene. The suspect is still outstanding.
The male suspect is described as being 6' to 6'2" tall with an average build. He was wearing a gray polo over a long-sleeved white shirt, dark pants, dark-colored neck gaiter, gloves, sunglasses, and a green ball cap.
This case is still under investigation.
There were no reported injuries. There is no further information at this time.
The Yuma Police Department encourages anyone with any information about this case to please call the Yuma Police Department at 928-373-4700 or 78-Crime at (928) 782-7463 to remain anonymous.


text:
Payz at Online Casinos
Payz is an e-wallet based payment service that offers customers a safe, instant, and convenient way of sending and receiving money worldwide, all in a single online account.
Besides a mobile application, it also includes a prepaid contactless debit card, which provides direct access to funds stored in the user´s account and allows for in-store purchases, as well as cash withdrawals at ATMs.
For years, Payz ↱ has successfully provided end-to-end payment solutions to individuals and merchants across 150 countries, including India, and in 45 currencies.
Top India casinos with Payz in 2024
The Payz e-wallet grants users the freedom to carry out secure online transactions without worrying about their privacy.
This payment ecosystem employs the latest security and anti-fraud technology, giving user´s a quick and safe way of transferring funds to and from other consumers and associated partners.
Such has been the company´s success that it is now a global presence, with its services and operations expanding to other markets and distant countries.
Indeed, many online sportsbooks and casinos in India support Payz for deposits and withdrawals, some of which you can find listed on this page.
All of our recommended gambling sites have been tested and reviewed by a team of experts. Moreover, they are fully licensed—some even in European jurisdictions—and enjoy a good reputation amongst the public.
We also accompany our list of top Payz casinos with a review of each one detailing its web design, games, bonuses, terms and conditions, and other relevant aspects.
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(T&Cs apply)150% up to ₹15,000 + ₹101 Daily Cashback for 7 days ▼ More Details ▼ 18+. New customers. Opt-in required. 150% Deposit Bonus : 150% up to ₹15,000. Min Dep ₹1,000. ₹101 Daily Cash back : ₹101 daily for 7 Days. Min dep ₹1,500. 30x bonus wagering (game weighting applies). T&Cs apply. |
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(T&Cs apply)no welcome bonus available, 10% Cashback instead ▼ More Details ▼ *18+, all players, 10% lifetime cashback on losses within a day when balance drops to zero or below £10, Cashback is credited the following day and can be cashed out or played, cashback is valid for 10 days, T&Cs apply |
*Bonus T&Cs apply! Accuracy not guaranteed, terms can change any time.
Table of contents:
How does Payz work?
With the Payz digital wallet, users can make international money transfers in multiple currencies without setup fees.
To use the payment service, you will need to open an account, which is free of charge, easy, and only takes a few minutes.
All you have to do is create a username and a password and fill in a short signup form with your basic details, including name, birth date, email address, and telephone number.
You are not even required to have a bank account, and the company does not run any credit checks.
Once registered, you can send funds instantly and for low rates, although not all of the wallet´s features will be available.
So, to make full use of the account, you must verify your identity by providing a copy of your ID card, driver´s license, or passport, in addition to proof of address in the form of a utility bill, a bank statement, or a certificate of residence.
The entire verification process can take from a few minutes to several days, and as soon as you complete it, all services and functions will be available to you.
These include multi-currency accounts, higher purchase limits, free international transfers, four Mastercards, and a one-time-use virtual prepaid card.
Before making an online payment, be sure to top up your ecoAccount with money, for which you can use different methods, such as credit or debit cards and bank transfers.
It should be noted that the Payz account has five membership levels you can choose from: classic, silver, gold, platinum and VIP – each with its own fees and perks.
Advantages and disadvantages of Payz at online casinos
✔ | Easy to use platform |
✔ | Quick online payments |
✔ | Prepaid debit card available |
✔ | Accepted at many gambling sites |
✔ | Supports 45 currencies, including INR |
✔ | Withdrawals are possible |
✖ | Certain fees may apply |
✖ | Multiple account limitations |
Casino deposits with Payz
After registering and topping your ecoAccount, you can immediately start using it to send, receive, and store money online.
In the case of casinos, funding the player account is a quick and straightforward process: just go to the "Cashier" section of the operator´s website, choose Payz as the payment method and introduce the deposit amount.
Thereafter, confirm the transaction. The money will appear in your wallet within seconds of the payment´s approval.
Casino withdrawals with Payz
Many online casinos also allow you to withdraw funds with Payz, and the process is similar to making a deposit.
Once more, visit the "Cashier" section of the site, select the e-wallet as a payment option, enter the withdrawal amount, and submit the request.
However, unlike the deposit, this transaction is not instant; most likely taking between two to three business day, as the casino has to review your request and check your identity before processing the payment.
Our verdict
In short, Payz is a safe, flexible, and convenient payment service that lets users send and receive money worldwide.
Apart from shopping on the Internet, you can use the e-wallet to make deposits and withdrawals at many online casinos.
And like other similar methods, it is possible to pay without disclosing your financial details.
One drawback, however, is the fees Payz charges for some of its wallet´s services, but do not fret: the prices are reasonably fair.
Remember that your ecoAccount is part of an ecosystem of products that includes a physical Mastercard and virtual prepaid debit card.
Payz FAQ
The Payz account supports a variety of fiat currencies, such as the Indian rupee, the US dollar, the British pound, and the euro.
It is also compatible with the Turkish lira, the Argentine peso, the Australian dollar, the Bosnian mark, the Bulgarian lev, the Brazilian real, the Canadian dollar, the Chilean peso, the Chinese yuan renminbi, the Colombian peso, the Costa Rican colon, the Czech koruna, the Denmark krone, the Georgian Lari, the Hong Kong Dollar, the Croatian kuna, the Hungarian forint, the Indonesian rupiah, the Israeli new shekel, the Icelandic króna, the Japanese yen, the Kenyan shilling, the Moldovan lei, the Macanese pataca, the Mexican peso, the Malaysian ringgit, the Nigerian naira, the Nicaraguan córdoba, the Norwegian kroner, the New Zealand dollar, the Panamanian balboa, the Peruvian sol, the Philippine pesos, the Polish zloty, the Romanian leu, the Russian rubble, the Rwandan franc, the Serbian dinar, the Singapore dollar, the the South African rand, Swedish kroner, the Swiss franc, the Thai baht, the Tanzanian shilling, the Ukrainian hryvnia, the Uzbekistani so´m, and the Central African franc.
While opening an ecoAccount is free of charge, some of the services provided by the e-wallet are not.
For example, there are different types of transactions that carry fees, all of which you can find on the Payz website; and depending on your membership level, you can lower them.
Overall, the wallet´s fee structure is competitive, and its prices are not that high, considering the quality of the service you are receiving.
Bear in mind that some casino operators apply charges to deposits or withdrawals and add them to the payment provider´s fees.
Funds sent to a player account through Payz are credited instantly, provided the user has topped the e-wallet before making the deposit.
Withdrawals, however, might take a bit longer, as the casino will first have to verify your account.
Remember that each operator has a different processing time for withdrawal requests and that they will only offer Payz as an option if you previously used it for the deposit.
There are plenty of online and mobile casinos that admit Payz as a payment method for deposits and withdrawals. You can even find a list of the top ones here, in Casino Daemon India.
The minimum and maximum limits for deposits depend on the casino´s payment policy; in general, however, you can deposit anywhere from $10 to $1,000 per transaction.
As for withdrawals, the minimum limits are usually similar to those for deposits, but the maximum can vary greatly.
Some operators also apply limits to cumulative withdrawals on a daily, weekly or monthly period.
Yes. If a casino operator accepts payments made with Payz, it will most likely offer its players bonuses for making deposits with this e-wallet.
Nonetheless, read the casino´s terms and conditions, as some sites exclude specific methods from the bonus money.
As a regulated e-wallet, Payz meets the highest security standards of the payment industry, which is no surprise since protecting the clients´ financial data is its top priority.
Through advanced security measures, Payz adds to your account an extra layer of protection against viruses, spyware, and cyberattacks.
All transactions made with the account, for instance, are automatically encrypted from your browser to the provider´s website using either the Secure Sockets Layer (SSL) protocol or its successor, the Transport Layer Security (TLS).
Furthermore, servers that contain private information are physically separated from the rest of the hardware in highly restricted and secure areas.
To ensure the safety of your money, the fintech company behind the wallet also follows a procedure known as safeguarding, a requirement for all electronic money institutions.
This process obliges Payz to separate its funds from the users´ in a safeguarding account.
The Payz digital wallet offers players a convenient and safe way of depositing and withdrawing funds at many online casinos.
Aside from the ecoAccount, you can also obtain a physical Mastercard and a virtual prepaid card with which you can access the wallet´s balance and make payments.


text: There are good doctors….And there are excellent human beings as good doctors….Dr Himanshu Matalia belongs to the latter category My mother,70 yrs old fragile lady,suffering with multiple health issues and has visited hundreds of doctors for various ailments but none…NONE was as humble and empathetic as Dr Himanshu was.She got operated for her right eye cataract in September 2020,she came out smiling from.the OT exclaiming….I can see all so clearly . Then on 25th Feb…Yesterday…She got operated for her left eye.Me and my mom travelled from Delhi specially for Dr Himanshu despite the fact that many of my batchmates are great doctors in various hospitals in Delhi ,including AIIMS.But she chose only Dr Himanshu and I'm so happy I came here .The way Dr Himanshu speaks,calming an already agitated and troubled soul… No one I've seen can do.I learnt so much from him despite being in the same profession.Thank you Dr Himanshu and his team for the wonderful service you are giving to the society…This world needs more people like you!!God bless you


text:
"I like the flavor from smoke, but at the same time I enjoy the convenience of electrical equipment." That's Paul Qui's take on his dream kitchen, which he describes to Digital Trends in the midst of the SouthBites food truck frenzy downtown: "With the technology these days, for me, it's not about fire to bring out flavors; there are other ways to extract flavors from things." [Digital Trends]
Filed under:


text:
The human brain holds many clues about a person's long-term health.
In fact, research shows that a person's brain age is a more useful and accurate predictor of health risks and future disease than their birthdate.
In a study from the University of Southern California, scientists developed a new artificial intelligence (AI) model that analyzes magnetic resonance imaging (MRI) brain scans.
It could be used to accurately capture cognitive decline linked to neurodegenerative diseases like Alzheimer's much earlier than previous methods.
Brain aging is considered a reliable biomarker for neurodegenerative disease risk. Such risk increases when a person's brain exhibits features that appear "older" than expected for someone of that person's age.
In the study, the team used the deep learning capability of the novel AI model to analyze the scans, and they detected subtle brain anatomy markers that are otherwise very difficult to detect and that correlate with cognitive decline.
The team collated the brain MRIs of 4,681 cognitively normal participants, some of whom went on to develop cognitive decline or Alzheimer's disease later in life.
Using these data, they created an AI model called a neural network to predict participants' ages from their brain MRIs.
First, the researchers trained the network to produce detailed anatomic brain maps that reveal subject-specific patterns of aging.
They then compared the perceived (biological) brain ages with the actual (chronological) ages of study participants. The greater the difference between the two, the worse the participants' cognitive scores, which reflect Alzheimer's risk.
The results show that the model can predict the true (chronological) ages of cognitively normal participants with an average absolute error of 2.3 years.
The new model also showed sex-specific differences in how aging varies across brain regions. Certain parts of the brain age faster in males than in females, and vice versa.
Males, who are at higher risk of motor impairment due to Parkinson's disease, experience faster aging in the brain's motor cortex, an area responsible for motor function.
Findings also show that, among females, typical aging may be relatively slower in the right hemisphere of the brain.
The team says the earlier doctors can identify people at high risk for Alzheimer's disease, the earlier clinicians can intervene with treatment options, monitoring, and disease management.
What makes AI especially powerful is its ability to pick up on subtle and complex features of aging that other methods cannot and that are key in identifying a person's risk many years before they develop the condition.
If you care about Alzheimer's, please read studies about the root cause of Alzheimer's disease, and new non-drug treatment could help prevent Alzheimer's.
If you care about brain health, please read studies about how the Mediterranean diet could protect your brain health, and Strawberries could help prevent Alzheimer's disease.
The study was conducted by Andrei Irimia et al and published in Proceedings of the National Academy of Sciences.
Copyright © 2023 Knowridge Science Report. All rights reserved.


text: It is important to note that higher doses also increase the risk of side effects, including headaches and nausea. Patients should always follow their doctor's instructions and report any adverse reactions. In addition, Viagra 100 mg should not be taken more than once per day. As with any medication, it is important to weigh the potential benefits and risks with a healthcare provider


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We have updated our Privacy Policy Please take a moment to review it. By continuing to use this site, you agree to the terms of our updated Privacy Policy.
Julia Baird
Julia Baird is a journalist, broadcaster and author based in Sydney, Australia. She is a columnist for the International New York Times and host of The Drum on ABC (Australia). Her writing has appeared in Newsweek, the New York Times, Philadelphia Inquirer, Guardian, Washington Post, Sydney Morning Herald, Monthly and Harper's Bazaar. She has a PhD in history from the University of Sydney. In 2005, Baird was a fellow at the Joan Shorenstein Center on Media, Politics and Public Policy at Harvard University.
Read More


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The masks are "in fashion" at the Palacio de Santoña, in Madrid, where the chess Candidates tournament is held. The first days it was rare to see the hated 'mask', but there have been several cases of Covid, some in the players' environment, and precautions have been extreme. The majority of the public continues to go without protection, but it is increasingly normal to see journalists, photographers and members of the organization wearing it, especially those who have closer contact with the great masters.
The organization confirms three cases, two on the first day, asymptomatic, and another with symptoms but who refused to test positive for three days. Last Monday night the contagion was confirmed.
The greatest danger, of course, is that one of the eight participants becomes infected. In everyone's memory is what happened in the last edition of the Candidates tournament, in the Russian city of Yekaterinburg. In March 2020, FIDE decided to celebrate it, despite the risk it posed, with the uncontrolled pandemic on half the planet. Several players expressed their concern and one of them, the Azerbaijani Teimour Radjabov, went so far as to withdraw, as well as accusing the organizers of being irresponsible. When the competition had just passed its halfway point, the International Federation had to rectify and suspend the tournament, which took a year to resume.
In the 2002 Candidates the situation is less dramatic and there was never any doubt about holding the tournament. Naturally, however, possible complications have been foreseen and FIDE has published a page to answer the most frequent questions that may arise.
Dr. María Rubio is in charge of examining and doing the necessary tests if someone has symptoms. Not only the players, but also the press, volunteers and anyone related to the tournament.
On the other hand, in the pre-tournament technical meeting in which the players participated, the possibility of one of the players being infected was discussed. Unlike in Yekaterinburg, where the contagion would have ended the tournament for the affected, here the grandmasters approved a more supportive solution.
The decision adopted is that if a player is affected, he will be able to continue playing, although in a separate room, where the two chess players involved will wear a mask. "It could happen to anyone," was the general comment. And no one wants to be left out because of a disease that, except in extreme cases, is no longer so serious, at least among people as young as the participants in Madrid. The oldest are Radjabov, 35 years old, and Nakamura, 34.
FIDE ensures that "the organizers will provide all the necessary technical equipment." Likewise, the referees "will supervise the development of the games". It does not say it clearly, but it seems that it is even planned that if there is a contagion, the two grandmasters involved in that game can play separately. They would not use the same board and could face each other telematically, although in the same room.
The risk is greater if you take into account that many of the players, something worthy of applause, stop to take photos and sign autographs for fans at the entrance and exit of the game room. It usually occurs outdoors, but very few wear a mask.
Apart from all the measures mentioned, the tournament doctor takes the temperature of the players before each round, there are masks for everyone in different rooms of the Palace and the pieces and boards are disinfected before each game.


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Next page . . . . Previous page . . . . Speeches . . . . Contents . . . . Debates(HTML) . . . .
Legislative Assembly for the ACT: 2001 Week 8 Hansard (9 August) . . Page.. 2641 ..
MR SMYTH (continuing):
The legislation provides for tests to be used in assessing accreditation applications. This will allow the Road Transport Authority to apply 'fit and proper' tests to applicants for operator and network accreditation.
Under the provisions of the Bill, it will be at the discretion of the Road Transport Authority to audit operators and network providers to determine compliance with the accreditation requirements and to maintain service quality and public safety.
Similar to the approach adopted in the Road Transport (Public Passenger Services) Act 2001, the Amendment Bill provides that the powers, duties, conduct, training and attire of drivers may be regulated. The legislation also maintains the power of the Minister to determine maximum taxi fares.
Two provisions previously included in the taxi legislation in the Road Transport (General) Act 1999 have not been carried over to the Amendment Bill, based on recommendations of the National Competition Policy Review. These Items are the requirement for the Minister to set a reserve price for a taxi licence and the provision limiting the number of licences that may be held by a person to two.
The Road Transport (Public Passenger Services) Amendment Bill 2001 contains significant changes to the regulatory environment for taxi services and provides the framework for the provision of services that are responsive to community needs for safe, reliable and efficient public passenger taxi services.
Debate (on motion by Mr Hargreaves ) adjourned to the next sitting.
Stock Amendment Bill 2001
Mr Smyth , pursuant to notice, presented the bill and its explanatory memorandum.
Title read by Clerk.
MR SMYTH (Minister for Urban Services, Minister for Business, Tourism and the Arts and Minister for Police and Emergency Services) (10.59): I move:
That this bill be agreed to in principle.
I seek leave to have my tabling speech incorporated in Hansard.
Leave granted.
The speech read as follows:
This Bill will increase the coverage of material banned from being fed to ruminants. Just in case any members are uncertain, a ruminant is an animal with four stomachs and which chews its cud. Sheep, cattle and goats are the obvious examples.
There is an unproven link between bovine spongiform encephalopathy known as BSE or 'Mad Cow Disease' and the variant form of Creutzfeld-Jakob Disease, which affects humans.
Next page . . . . Previous page . . . . Speeches . . . . Contents . . . . Debates(HTML) . . . .


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Cuvée des Cadettes 2018
Cuvée des Cadettes 2018
AOP Châteauneuf-du-Pape
The Cuvée des Cadettes is an entire story!
Indeed, it was the first plot replanted after the phylloxera aphid ravaged millions of hectares of vines. We owe this first plantation to Joseph Ducos, the estate's owner, in the nineteenth century. Since then, the Cuvée des Cadettes has been made with the best grenache, syrah, and mourvèdre grapes expressing this unique terroir – elegant, intense, sophisticated, and well-balanced. To be enjoyed with wild game, aged meats, and other dishes as rare and complex as this wine.
Purple color with shimmering hints. Lots of aromas on the nose: wild blackberries, ripe cherries along with vanilla and dried -owers notes. Rich and opulent on the palate, this wine is impressive, it delivers -avors of jammy red berries, sweet spices (cinnamon) with some herbal touches (men-thol). It is highly concentrate and a little smoky. The nish is long with solid but ne tannins. A great wine to keep a decade or more!


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This is a series of 1.5 week exercises in which we designed 1,200 square feet homes. These quick exercises were to help us understand and draw smaller scale living spaces before moving on to designing a large-scale 60+ unit collective housing development.
First was a warm-up drawing exercise in which I chose to draw a Nicholson 32 sailboat. Then was the horizontal home which was required to be a single story with 25% outdoor courtyard space. Next was the vertical home which was to have a 250 SF footprint, then grow upward. Lastly, was the sectional home which was to have the same footprint, but then grow up and out, sort of like a mushroom.


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The first ever professional cordless/corded super torque Skin clipper debulks down to skin in record time! This clipper takes hair to the skin like a trimmer but has the powerful motor and size of a clipper! Having the ability to debulk large amounts of hair saves time and prolongs the life of a trimmer. The super torque motor runs at 7,200 RPMS and is fine-tuned for increased efficiency and performance.
The Skin Clipper is engineered with a Lithium-ion battery that delivers a 3-hour run-time and can fully charge in 150 minutes. Recharge the Skin Clipper 3 different ways; with the universal micro-USB cord, 2-prong pass-through charging cord, or the included charging stand. The LED battery light indicator on the front will activate when charging, once charging is complete the light will deactivate.
Italian designed and engineered for peak performance. Introducing new color series in matte metallic green, matte gold, and matte gunmetal. A convenient hook located on the bottom is great for hanging and storage when not in use. This clipper is made to be used without a lever, conventional bracket kit will be available to add a lever and modify the Skin into a conventional style clipper (kit sold separately).
Gamma+ has collaborated with TaylorCutz, a giant in the barber industry with over 25 years of experience. Taylor has become a point of reference for many professional barbers and worked closely with Gamma+ to perfect the Skin clipper. After a rigorous process of testing and adjusting he has officially given the Skin his stamp of approval. His fading and cutting techniques inspire the masses and set global trends.
Assembled with a fixed Black Diamond Carbon DLC SKIN blade with super fine, shallow teeth that smoothly debulk no matter the hair type. The Black Diamond's deep slim moving blade feeds hair efficiently and smoothly to prevent snagging and pulling when cutting large amounts of hair. Thick, coarse, thin, or fine hair - the Skin provides the quickest, most efficient cut.
INCLUDES: 1 Year Warranty, Balding Clipper, Double Black Diamond Carbon DLC Skin balding blade, Deep Slim moving blade, 5 black guard attachments (1/16", 1/8", 1/4", 3/8", 1/2"), micro-USB cord, charging stand, maintenance kit.


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Samsung Galaxy A35 5G is expected to launch in the market soon. The phone is likely to succeed the Samsung Galaxy A34 5G, which was unveiled this year in March with an IP67 rating. The launch timeline of the succeeding smartphone has not been tipped yet. The company has also not given out any details on the Galaxy A35. However, ahead of any official announcements, design renders of the Galaxy A35 and some of its key specifications have surfaced online.
A MySmartPrice report has shared leaked renders and a 360-degree video showing the design of the purported Samsung Galaxy A35 5G model. The phone is seen with rounded corners, a flat display with slim side and top bezels, and a relatively thick chin. There is a centre-aligned hole-punch slot at the top of the panel for the front camera. The screen is said to be 6.6-inch in size.
Leaked Samsung Galaxy A35 5G render
Photo Credit: MySmartPrice/Onleaks
The back panel of the Galaxy A35 5G appears to have a triple rear camera unit placed in a similar vertical alignment as seen on the preceding Galaxy A34 model and many other 2023 Samsung models. The three camera units are placed in vertically arranged separate circular slots in the top left corner. The brand name is engraved towards the base.
Further, the right edge of the Galaxy A35 5G is seen with a slight bump where the volume rocker and power button are placed. The report adds that the handset is likely to measure 161.6mm x 77.9mm x 8.2mm in size. If the frame bump is taken into account, the handset is said to measure 78.5mm in width. No other specifications about the smartphone were hinted at in the report. More details can be expected closer to the launch of the handset.
Samsung's Galaxy A34 5G was launched in India in Black, Light Green, Light Violet, and Silver colour options. The 6GB + 128GB of the phone is listed at Rs. 26,999, while the 8GB + 128GB and 8GB + 256GB variants are priced at Rs. 28,999 and Rs. 30,999, respectively.
Samsung launched the Galaxy Z Fold 5 and Galaxy Z Flip 5 alongside the Galaxy Tab S9 series and Galaxy Watch 6 series at its first Galaxy Unpacked event in South Korea. We discuss the company's new devices and more on the latest episode of Orbital, the Gadgets 360 podcast. Orbital is available on Spotify, Gaana, JioSaavn, Google Podcasts, Apple Podcasts, Amazon Music and wherever you get your podcasts.
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For the latest tech news and reviews, follow Gadgets 360 on X, Facebook, WhatsApp, Threads and Google News. For the latest videos on gadgets and tech, subscribe to our YouTube channel.
Realme GT 5 Pro Teased to Pack 50-Megapixel Sony IMX890 Camera With 3x Optical Zoom