Attenuated regenerative properties in human periodontal ligament-derived stem cells of older donor ages with shorter telomere length and lower SSEA4 expression

Periodontal ligament (PDL) stem cell properties are critical in the periodontal tissue regeneration for periodontitis. Previously, we have demonstrated that cigarette smoking attenuates PDL-derived stem cell (PDLSC) regenerative properties. Here, we report the findings on the regenerative properties of human PDLSCs with different donor ages and the underlying mechanisms. Human PDLSCs from 18 independent donors were divided into different age groups (≤ 20, 20-40, and > 40 years old). The proliferation of PDLSCs with donor age of ≤ 20 years old was significantly higher than that of the 20-40- and > 40-years-old groups, whereas the migration of PDLSCs with donor age of ≤ 20 and 20-40 years old was significantly higher than that of the > 40-years-old group. Moreover, the mesodermal lineage differentiation capabilities of PDLSCs were also higher in the donor age group of ≤ 20 years old than the donor age of > 40 years old. In addition, shorter telomere length and lower expression of SSEA4 were found in PDLSCs with donor age of > 40 years old, compared with those with donor age of ≤ 20-years-old group. Besides, PDLSCs with donor age of 20-40 and > 40 years old had higher IL6 and CXCL8 gene expressions. In summary, results from this study revealed the attenuated proliferation, migration, and mesodermal lineage differentiation properties in human PDLSCs with older donor ages. Donor age of PDLSCs should be considered as the selection criteria for the periodontal tissue regeneration treatment

Supplementary document for Glucose sensing based on hydrogel grating incorporating phenylboronic acid groups - 6162183.pdf

The FTIR spectroscopy of polyacrylamide hydroge

Mutations of RagA GTPase in mTORC1 Pathway Are Associated with Autosomal Dominant Cataracts.

Cataracts are a significant public health problem with no proven methods for prevention. Discovery of novel disease mechanisms to delineate new therapeutic targets is of importance in cataract prevention and therapy. Herein, we report that mutations in the RagA GTPase (RRAGA), a key regulator of the mechanistic rapamycin complex 1 (mTORC1), are associated with autosomal dominant cataracts. We performed whole exome sequencing in a family with autosomal dominant juvenile-onset cataracts, and identified a novel p.Leu60Arg mutation in RRAGA that co-segregated with the disease, after filtering against the dbSNP database, and at least 123,000 control chromosomes from public and in-house exome databases. In a follow-up direct screening of RRAGA in another 22 families and 142 unrelated patients with congenital or juvenile-onset cataracts, RRAGA was found to be mutated in two unrelated patients (p.Leu60Arg and c.-16G>A respectively). Functional studies in human lens epithelial cells revealed that the RRAGA mutations exerted deleterious effects on mTORC1 signaling, including increased relocation of RRAGA to the lysosomes, up-regulated mTORC1 phosphorylation, down-regulated autophagy, altered cell growth or compromised promoter activity. These data indicate that the RRAGA mutations, associated with autosomal dominant cataracts, play a role in the disease by acting through disruption of mTORC1 signaling

Agonist of growth hormone–releasing hormone enhances retinal ganglion cell protection induced by macrophages after optic nerve injury

Optic neuropathies are leading causes of irreversible visual impairment and blindness, currently affecting more than 100 million people worldwide. Glaucoma is a group of optic neuropathies attributed to progressive degeneration of retinal ganglion cells (RGCs). We have previously demonstrated an increase in survival of RGCs by the activation of macrophages, whereas the inhibition of macrophages was involved in the alleviation on endotoxin-induced inflammation by antagonist of growth hormone–releasing hormone (GHRH). Herein, we hypothesized that GHRH receptor (GHRH-R) signaling could be involved in the survival of RGCs mediated by inflammation. We found the expression of GHRH-R in RGCs of adult rat retina. After optic nerve crush, subcutaneous application of GHRH agonist MR-409 or antagonist MIA-602 promoted the survival of RGCs. Both the GHRH agonist and antagonist increased the phosphorylation of Akt in the retina, but only agonist MR-409 promoted microglia activation in the retina. The antagonist MIA-602 reduced significantly the expression of inflammation-related genes Il1b, Il6, and Tnf. Moreover, agonist MR-409 further enhanced the promotion of RGC survival by lens injury or zymosan-induced macrophage activation, whereas antagonist MIA-602 attenuated the enhancement in RGC survival. Our findings reveal the protective effect of agonistic analogs of GHRH on RGCs in rats after optic nerve injury and its additive effect to macrophage activation, indicating a therapeutic potential of GHRH agonists for the protection of RGCs against optic neuropathies especially in glaucoma

<i>RRAGA</i> mutations identified from congenital or juvenile-onset cataract patients.

<p>(<b>A</b>) The genomic features of the <i>RRAGA</i> gene and the domains in the encoded protein. <i>RRAGA</i> contains a single exon encoding a GTP-binding domain (red) and a C-terminal domain (yellow). The UTR regions are shown in green. Frequencies of mutations and patients with mutations are indicated as numbers in blue filled circles. The p.Leu60Arg mutation was observed in affected of Family 1 and unrelated patient CC38, while c.-16G>A was found in unrelated patient CC19. (<b>B</b>) Protein sequence alignment of RRAGA orthologs in vertebrates. The switch I and II regions in the GTP-binding domain are shaded purple [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006090#pgen.1006090.ref012" target="_blank">12</a>]. The conserved leucine residue at codon 60 mutated in affected patients, indicated by a red triangle, is located within switch II. (<b>C</b>) Protein homology modeling of the GTP-binding domain of RRAGA. The GTP molecule is shown in purple, and the Mg²⁺ ion in orange. The L60 residue is within the switch II region responsible for mTORC1 activation [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006090#pgen.1006090.ref012" target="_blank">12</a>].</p

The c.-16G>A mutation in the 5’-UTR of <i>RRAGA</i> is associated with reduced promoter activity.

<p>(<b>A</b>) The 5’-UTR and start codon of <i>RRAGA</i>. The start codon (ATG) is shown in green. The <i>RRAGA</i> 5’-UTR is predicted to be associated with promoter activity (Ensembl feature: ENSR00001469646) in the Ensembl database, and is enriched with CpG dinucleotides (highlighted in blue). The c.-16G>A mutation indicated by a red triangle, overlaps with a CpG dinucleotide and a predicted binding site for transcription factor E2F1, which regulates mTORC1 signaling [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006090#pgen.1006090.ref017" target="_blank">17</a>]. The mutant A allele was predicted by PROMO [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006090#pgen.1006090.ref016" target="_blank">16</a>] to abolish the binding site. (<b>B</b>) The c.-16G>A mutant is associated with reduced luciferase reporter expression compared with the wild-type (WT) in B-3 cells. B-3 cells were transfected with pGL3-enhancer vectors with no RRAGA 5’-UTR (VT), or with the wild-type RRAGA 5’-UTR (WT), or 5’-UTR c.-16G>A mutant. The expression level of luciferase reporter was determined using immunoblotting. (<b>C</b>) Quantification of the immunoblotting results of luciferase expression. Data are presented as the mean plus S.E.M. <i>P</i> values were determined by Student's t test. *, <i>P</i> < 0.05.</p

Clinical features of congenital or juvenile-onset cataract patients with <i>RRAGA</i> mutations in the current study.

<p>Clinical features of congenital or juvenile-onset cataract patients with <i>RRAGA</i> mutations in the current study.</p

Effect of the p.Leu60Arg mutation in human lens epithelial cells.

<p>(<b>A-F</b>) Live imaging of B-3 cells transfected with the GFP vector (VT), GFP-RRAGA wild-type (WT) or p.Leu60Arg mutant (Leu60Arg). Arrow denotes reduced cell area of in cells expressing Leu60Arg. Scale bar indicates 250 μm. (<b>G</b>) GFP-trap immunoprecipitates (IP) from transfected B-3 cells analyzed by immunoblotting using antibody against GFP. Results of total cytosol (input), unbound protein and immunoprecipitated (bound) protein are shown. GAPDH is used as a loading control. (<b>H-O</b>) Co-transfection of RFP-LAMP1 with GFP-RRAGA in B-3 cells. Arrows denote co-localization between RRAGA Leu60Arg and LAMP1. RRAGA Leu60Arg was mostly located within the lysosomes. Scale bar indicates 10 μm. (<b>P</b>) Immunoblotting of mTOR, p-mTOR (Ser2448), and the autophagy marker LC3B in B-3 cells expressing GFP, or RRAGA WT or Leu60Arg. (<b>Q, R</b>) Quantification of the immunoblotting results of mTOR and LC3B-II/LC3B-I ratio. The phosphorylated form, p-mTOR (Ser2448) was upregulated in cells transfected with RRAGA Leu60Arg than those with RRAGA WT. In line with such mTOR changes, a remarkable reduction of LC3B-II/LC3B-I ratio was found in cells expressing RRAGA Leu60Arg compared to those expressing RRAGA WT. Data are presented as the mean plus S.E.M. <i>P</i> values were determined by Student's t test. (<b>S</b>) Measurement of the area of B-3 cells expressing GFP (analyzed cell n = 183), or GFP-RRAGA WT (analyzed cell n = 158) or RRAGA Leu60Arg (analyzed cell n = 463). Mean plus S.E.M. is shown. <i>P</i> values were determined by the Mann-Whitney U test. *, <i>P</i> < 0.05.</p