Table_1_Identification of Key Biomarkers and Pathways for Maintaining Cognitively Normal Brain Aging Based on Integrated Bioinformatics Analysis.DOCX

BackgroundGiven the arrival of the aging population has caused a series of social and economic problems, we aimed to explore the key genes underlying cognitively normal brain aging and its potential molecular mechanisms.MethodsGSE11882 was downloaded from Gene Expression Omnibus (GEO). The data from different brain regions were divided into aged and young groups for analysis. Co-expressed differentially expressed genes (DEGs) were screened. Functional analysis, protein–protein interaction (PPI) network, microRNA (miRNA)-gene, and transcription factor (TF)-gene networks were performed to identify hub genes and related molecular mechanisms. AlzData database was used to elucidate the expression of DEGs and hub genes in the aging brain. Animal studies were conducted to validate the hub genes.ResultsCo-expressed DEGs contained 7 upregulated and 87 downregulated genes. The enrichment analysis indicated DEGs were mainly involved in biological processes and pathways related to immune-inflammatory responses. From the PPI network, 10 hub genes were identified: C1QC, C1QA, C1QB, CD163, FCER1G, VSIG4, CD93, CD14, VWF, and CD44. CD44 and CD93 were the most targeted DEGs in the miRNA-gene network, and TIMP1, HLA-DRA, VWF, and FGF2 were the top four targeted DEGs in the TF-gene network. In AlzData database, the levels of CD44, CD93, and CD163 in patients with Alzheimer’s disease (AD) were significantly increased than those in normal controls. Meanwhile, in the brain tissues of cognitively normal mice, the expression of CD44, CD93, and CD163 in the aged group was significantly lower than those in the young group.ConclusionThe underlying molecular mechanisms for maintaining healthy brain aging are related to the decline of immune-inflammatory responses. CD44, CD93, and CD 163 are considered as potential biomarkers. This study provides more molecular evidence for maintaining cognitively normal brain aging.</p

Predicted Primary Amino Acid Sequence of EAK-6 and Similarity with PTPs

<div><p>(A) EAK-6 amino acid sequence. EAK-6 sequence was derived from full-length cDNA amplified by RT-PCR from wild-type C. elegans total RNA. The PTP domain is denoted in boldface. The residue mutated in <i>eak-6(mg329)</i> is boxed and the predicted change indicated above the mutated residue. Amino acids encoded by an alternatively spliced exon present in EAK-6L but not in EAK-6S (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020099#s4" target="_blank">Materials and Methods</a>) are underlined.</p> <p>(B) EAK-6 homology with PTPs. The PTP domain of EAK-6 is aligned with that of SDF-9 and 3 human PTPs. Sequences used in the alignment are based on domains defined by Pfam [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020099#pgen-0020099-b101" target="_blank">101</a>] and correspond to amino acids 40 to 276 of PTPN1 (PTP1B), 57 to 308 of EAK-6, 31 to 283 of SDF-9, 265 to 500 of PTPRA (receptor-type PTPα), and 273 to 520 of PTPN11 (SHP-2). Alignment was performed using ClustalX 1.8 and MacBoxshade 2.15. Conserved and identical residues are shaded. Ten motifs conserved among 37 vertebrate PTPs [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020099#pgen-0020099-b068" target="_blank">68</a>] are underlined, and 19 residues that are invariant among 113 vertebrate PTP domains are boxed. The four invariant residues that are not identical in EAK-6 (R45, Q85, H214, and G220, numbered according to the PTPN1 primary sequence) are denoted with dots. The catalytic cysteine residue [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020099#pgen-0020099-b069" target="_blank">69</a>] is denoted by an asterisk. Two conserved residues that are not conserved in EAK-6, D181 and Q262, are denoted by arrowheads.</p></div

Dauer Formation Phenotypes of <i>eak</i> Mutants

<p><i>eak</i> single mutants, <i>eak-x;eak-y</i> double mutants, or <i>eak;akt-1</i> double mutants were assayed for dauer arrest at (A) 25 °C and (B) 27 °C. (C) <i>eak</i> 27 °C dauer arrest phenotypes are suppressed by a mutation in <i>daf-16/FoxO</i>. (D) <i>akt-1, eak-4,</i> and <i>eak-6</i> 27 °C dauer arrest phenotypes are suppressed by RNAi of <i>daf-16/FoxO</i> and <i>daf-12/NHR</i>. All error bars indicate standard deviation. All experiments were performed three times. Refer to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020099#pgen-0020099-st001" target="_blank">Table S1</a> for numbers of animals scored. <i>eak-5</i> is allelic to the synthetic dauer formation gene <i>sdf-9</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020099#pgen-0020099-b062" target="_blank">62</a>] and is referred to as <i>sdf-9</i> throughout the paper. Mutant alleles used were <i>daf-2(e1370), akt-1(mg306), eak-4(mg348), sdf-9(mg337)</i> and <i>sdf-9(ut187), eak-6(mg329),</i> and <i>daf-16(mgDf47)</i>. <i>sdf-9(ut187)</i> was used to construct the <i>eak-4;sdf-9, eak-6;sdf-9,</i> and <i>daf-16;sdf-9</i> double mutants. Multiple alleles of <i>eak-4</i> and <i>sdf-9</i> yielded phenotypes similar to those shown. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020099#pgen-0020099-st001" target="_blank">Table S1</a> for numbers of animals assayed.</p

Models of EAK-4, SDF-9, and EAK-6 Function

<div><p>(A) Cell autonomous signaling in XXX. A schematic of one XXX cell is shown. EAK proteins function in parallel with AKT-1 and in the same pathway as AKT-2 to promote nondauer development by potentiating DAF-9/CYP27A1 function either directly or indirectly. DAF-9/CYP27A1 synthesizes 3-keto-7(5α)-cholestenoic acid, a ligand that promotes reproductive development by inhibiting the nuclear hormone receptor DAF-12 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020099#pgen-0020099-b091" target="_blank">91</a>]. DAF-16/FoxO is denoted with dashed lines, since DAF-16A does not appear to be expressed in XXX. It is not known whether other DAF-16/FoxO isoforms or DAF-2/InsR are expressed in XXX.</p> <p>(B) Nonautonomous signaling from XXX. A schematic of the anterior portion of an animal is shown with the head pointing left. The pharynx is also shown. XXX cells are denoted by red ovals, and DAF-16/FoxO-expressing cells are denoted by green ovals. EAK proteins in the XXX cells generate signals that regulate the synthesis or secretion of a hormone that inhibits DAF-16/FoxO function in other cells.</p></div

Expression of AKT-1 in XXX is Sufficient to Rescue the 25 °C Dauer Phenotype of an <i>eak-4;akt-1</i> Double Mutant

<p><i>eak-4(mg326);akt-1(mg306)</i> double mutant animals carry a transgene containing the <i>akt-1</i> genomic region and 3' untranslated region under the control of the <i>eak-4</i> promoter. Three independent lines rescue <i>eak-4(mg326);akt-1(mg306)</i> dauer arrest at 25 °C. Error bars indicate standard deviation. This experiment was performed twice. Refer to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020099#pgen-0020099-st001" target="_blank">Table S1</a> for numbers of animals scored.</p

EAK-4 Amino Acid Sequence and Alignment with Four C. elegans Homologs

<p>Sequences represent the entire predicted amino acid sequences of all five genes. EAK-4 sequence was derived from full-length cDNA amplified by RT-PCR from wild-type C. elegans total RNA. Alignment was constructed using ClustalX 1.8 and MacBoxshade 2.15. Shaded residues indicate amino acid identity and conservation. Mutated residues in three alleles of <i>eak-4</i> are boxed, and the predicted amino acid change is indicated above the box. The conserved G at residue 2 and S/T/A (boxed) at residue 6 in the <i>N</i>-myristoylation consensus sequence [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020099#pgen-0020099-b070" target="_blank">70</a>] are denoted by an asterisk and a vertical arrow, respectively.</p

EAK-4, SDF-9, and EAK-6 Localize to the Plasma Membrane of the XXX Cells

<div><p>(A) <i>eak-4, sdf-9,</i> and <i>eak-6</i> promoters drive expression in the same cells. Wild-type animals harboring an extrachromosomal array with <i>eakp::GFP</i> and <i>sdf-9p::RFP</i> constructs were analyzed by fluorescence microscopy. Representative photographs are shown. Animals are oriented anterior left and dorsal up.</p> <p>(B) EAK-4::GFP, SDF-9::GFP, and EAK-6::GFP fusion proteins localize to the plasma membrane of XXX. Animals harboring EAK::GFP translational fusion constructs and an integrated <i>sdf-9p::RFP</i> array were analyzed using fluorescence microscopy. Representative photographs of a XXX cell are shown.</p> <p>(C) Mutation of the invariant glycine in the <i>N</i>-myristoylation motif of EAK-4 abrogates plasma membrane localization. Animals harboring either a wild-type EAK-4::GFP construct or an EAK-4::GFP construct with the glycine at position 2 mutated to alanine (G2A) were analyzed using fluorescence microscopy.</p></div

A Functional DAF-16A::GFP Fusion Protein Is Not Expressed in XXX

<p>Wild-type animals harboring integrated DAF-16A::GFP and <i>sdf-9p</i>::RFP arrays were analyzed by confocal microscopy. Representative photographs of a single animal are shown. The animal is oriented anterior left and dorsal up. Merging of GFP and RFP images reveals no colocalization of fluorescent proteins.</p

Proton and Copper Binding to Humic Acids Analyzed by XAFS Spectroscopy and Isothermal Titration Calorimetry

Proton and copper (Cu) binding to soil and lignite-based humic acid (HA) was investigated by combining X-ray absorption fine structure (XAFS) spectroscopy, isothermal titration calorimetry (ITC), and nonideal-competitive-adsorption (NICA) modeling. NICA model calculations and XAFS results showed that bidentate and monodentate complexation occurred for Cu binding to HA. The site-type-specific thermodynamic parameters obtained by combining ITC measurements and NICA calculations revealed that copper binding to deprotonated carboxylic-type sites was entropically driven and that to deprotonated phenolic-type sites was driven by entropy and enthalpy. Copper binding to HA largely depended on the site-type and coordination environment, but the thermodynamic binding mechanisms for Cu binding to the specific site-types were similar for the different HAs studied. By comparing the site-type-specific thermodynamic parameters of HA–Cu complexation with those of low molar mass organic acids, the Cu coordination could be further specified. Bidentate carboxylic–Cu complexes made the dominating contributions to Cu binding to HA. The present study not only yields molecular-scale mechanisms of ion binding to carboxylic- and phenolic-type sites of HA but also provides the new insight that the universal nature of site-type-specific thermodynamic data enables quantitative estimation of the binding structures of heavy metal ions to humic substances

Longitudinal Impact on Quality of Life for School-aged Children with Amblyopia Treatment: Perspective from Children

<p><i>Background</i>: To evaluate the longitudinal impact on health-related quality of life (HRQOL) during amblyopia treatment for school-aged children from children’s perspective.</p> <p><i>Methods</i>: School-aged children prescribed amblyopia treatment for the first time were recruited into the current study. Using a questionnaire, subjects’ HRQOL was assessed before patching treatment, and at 8 weeks and 16 weeks after the commencement of patching treatment. Evaluation of visual function and psychosocial aspect was included in the questionnaire. Visual acuity and demographic data of the subjects were recorded.</p> <p><i>Results</i>: Forty-four children, aged 7–12 years, with anisometropic amblyopia were included in the study. Visual acuity in the amblyopic eye improved 1.90 (0.41–3.74) and 3.98 (2.22–5.11) lines at follow-up weeks 8 and 16, respectively. Both the total score and subscales of the questionnaire were reduced at the first follow-up and recovered at the second follow-up. Scores at week 16 were higher than those before treatment in the psychosocial aspect (<i>p</i> = 0.003), and lower in the visual function aspect (<i>p</i> < 0.001), without significant difference in total score (<i>p</i> = 0.207). Visual acuity in the amblyopic eye and psychosocial expectations for treatment were the most important factors that influenced HRQOL during treatment.</p> <p><i>Conclusions</i>: From the children’s perspective, the impacts on visual function and psychosocial aspect were significant in the first two months of treatment, and could be adapted during therapy for school-aged children. More attention should be paid to negative effects of treatment on daily life and study at the stage of amblyopia treatment for school-aged children. Meanwhile, necessary precautions should be taken to help reduce the impacts.</p