mRNA expression analysis of the <i>SLC20A2</i> mutation.

<p>(A) Sequencing showing an imbalance of the c.510delA (p.R172fsX19) mutant and wild-type alleles in cDNA templates. (B) Relative quantity (mean ± SD) of <i>SLC20A2</i> transcripts derived from real-time quantitative polymerase chain reactions in 3 affected individuals and a normal individual.</p

Study subjects and imaging evaluation.

<p>(A) Pedigree of family PJ-IBGC. The proband is indicated by an arrow. Filled symbols represent affected individuals, including both symptomatic (black) and asymptomatic (gray). (B) CT scan of the affected individuals. (C) MRS examination of a 4-year-old girl. I 1: a 54-year-old man; II 1: a 37-year-old woman; II:3: a 24-year-old man; III 1: an 11-year-old girl; III 2: a 4-year-old girl.</p

Identification of the c.510delA <i>SLC20A2</i> mutation.

<p>(A) Sequencing chromatogram showing the heterozygous c.510delA mutation in <i>SLC20A2</i> (right) and the wild type sequence (left). (B) Heteroduplex mobility assay of the 171 bp PCR product derived from all the affected individuals and some unrelated normal individuals. After denaturation (95°C), re-annealed reactions were run under non-denaturing conditions. M, 100 bp DNA marker; N, normal individuals. The bands were visualized by silver-staining. (C) Schematic diagram of the wild-type and mutant SLC20A2 proteins. Purple regions represent the L183–V483 fragment of SLC20A2, which are important for the Pi transportation activity. The blue circle indicates the mutated amino acid residue. Amino-acid residues of the novel C-terminal peptides in the p.R172fsX19 mutant are given with the 19 new residues in red. The structure model was drawn using TOPO2 software (<a href="http://www.sacs.ucsf.edu/TOPO2/" target="_blank">http://www.sacs.ucsf.edu/TOPO2/</a>).</p

Sequences and positions of the primers used for mutation analysis of <i>SLC20A2</i>.

<p>Genomic position of PCR primers corresponding to the Feb 2009 human genome reference sequence GRCh37.</p

microRNA-124 Inhibits Migration and Invasion by Down-Regulating ROCK1 in Glioma

<div><p>Background</p><p>The extraordinary invasive capability is a major cause of treatment failure and tumor recurrence in glioma, however, the molecular and cellular mechanisms governing glioma invasion remain poorly understood. Evidence in other cell systems has implicated the regulatory role of microRNA in cell motility and invasion, which promotes us to investigate the biological functions of miR-124 in glioma in this regard.</p><p>Results</p><p>We have found that miR-124 is dramatically downregulated in clinical specimen of glioma and is negatively correlated with the tumor pathological grading in the current study. The cells transfected by miR-124 expression vector have demonstrated retarded cell mobility. Using a bioinformatics analysis approach, rho-associated coiled-coil containing protein kinase 1 (ROCK1), a well-known cell mobility-related gene, has been identified as the target of miR-124. A dual-luciferase reporter assay was used to confirm that miR-124 targeted directly the 3′UTR of ROCK1 gene and repressed the ROCK1 expression in U87MG human glioma cell line. Furthermore, experiments have shown that the decreased cell mobility was due to the actin cytoskeleton rearrangements and the reduced cell surface ruffle in U87MG glioma cells. These results are similar to the cellular responses of U87MG glioma cells to the treatment of Y-27632, an inhibitor of ROCK protein. Moreover, a constitutively active ROCK1 in miR-124 over-expressed glioma cells reversed the effects of miR-124. Our results revealed a novel mechanism that miR-124 inhibits glioma cells migration and invasion via ROCK1 downregulation.</p><p>Conclusions</p><p>These results suggest that miR-124 may function as anti-migration and anti-invasion influence in glioma and provides a potential approach for developing miR-124-based therapeutic strategies for malignant glioma therapy.</p></div

ROCK1 is target of miR-124.

<p>(A) Illustration of the predicted miR-124-binding sequences in the 3′UTR region of ROCK1. (B) The calculated free energy for hybridization of the ROCK1 3′UTR and miR-124 (Red color: ROCK1, Green color: miR-124). (C) Homology analysis of the 3′UTR sequences of 13 different species recognized by miR-124 seed sequence. (D) Luciferase analysis in HEK293ET cells. The assay was repeated three times with each assay being performed in three wells, and similar results were obtained each time. (E) qRT-PCR assay of ROCK1 levels treated with either pcDNA3.1 or pcDNA3.1-miR-124 for 48 h, compared with mock control in U87MG cells. (F) Western blot analysis of ROCK1 expression treated with either pcDNA3.1 or pcDNA3.1-miR-124 for 72 h, compared with mock control in U87MG cells.</p

Proposed model of miR-124 function in glioma development and progression.

<p>The target of miR-124 in bold fonts was confirmed in this study.</p

qPCR assays of miR-124 expression levels in glioma tissue samples.

<p>The expression level of miR-124 was downregulated significantly in high grade human glioma tissues (five grade III and three grade IV) than that in low grade human glioma tissues (three grade I and five grade II) determined using qRT-PCR.</p

In silico development of a novel anti-mutation, multi-epitope mRNA vaccine against MPXV variants of emerging lineage and sub-lineages by using immunoinformatics approaches

Over the past year, an unexpected surge in human monkeypox (hMPX) cases has been observed. This outbreak differs from previous ones, displaying distinct epidemiological characteristics and transmission patterns, believed to be influenced by a newly emerging monkeypox virus (MPXV) lineage. Notably, this emerging MPXV lineage has exhibited several non-synonymous mutations, some of which are linked to immunomodulatory activities and antigenic characteristics that aid in host detection. However, specific treatments or vaccines for human monkeypox are currently lacking. Hence, we aim to develop a multi-epitope mRNA vaccine by using immunoinformatics approaches against the MPXV, particularly its emerging variants. Six proteins (A29L, A35R, B6R, M1R, H3L, and E8L) were chosen for epitope and mutation site identification. Seventeen top-performing epitopes and eight epitopes containing mutation sites were selected and combined with adjuvants, the PADRE sequence, and linkers for vaccine development. The molecular and physical properties of the designed vaccine (WLmpx) were favorable. Immunological characteristics of WLmpx were assessed through molecular docking, molecular dynamics (MD) simulations, and immune simulations. Finally, the vaccine sequence was utilized to formulate an mRNA-based vaccine. The informatics-based predicted results indicated that the designed vaccine exhibits significant potential in eliciting high-level humoral and cellular immune responses, but further validation through in vivo and vitro studies is warranted. Communicated by Ramaswamy H. Sarma</p

Identification of biological function of miR-124 in U87MG cells.

<p>(A) In vitro invasion assay. Invasive cells were stained and the average number of cells was counted at random six fields of vision. The data was an average value of three independent experiments. (Magnification: 100×; scale bars: 100 µm) (B) Stress fiber staining in U87MG glioma cells (Scale bars: 20 µm). (C) Scanning electron microscopy of U87MG cells treated with miR-124 expression vector or control and Y-27632 (Scale bars: 10 µm).</p