Normal pressure hydrocephalus, a possible complication in IgG4-related disease

We report a 75-year-old man with glucocorticoid-responsive normal pressure hydrocephalus (NPH) complicated with tubulointerstitial renal disease, protein-losing gastroenteropathy (PLG) and elevated serum IgG4, which might be a possible subset of IgG4-related disease (IgG4-RD). Although either PLG or NPH, especially in combination, has rarely been reported in IgG4-RD, the glucocorticoid-responsive nature of every abnormality observed in the patient supports the diagnosis of IgG4-RD. Of course, pathological confirmation is essential to fulfill the recently raised diagnostic criteria, however, such invasive procedure might not always be indicated in high-risk patients. Our report illustrates that IgG4-RD might be considered as one of the underlying causes and/or the aggravating factors of NPH before shunt operation

Effects of Angiotensin II on Erythropoietin Production in the Kidney and Liver

The kidney is a main site of erythropoietin production in the body. We developed a new method for the detection of Epo protein by deglycosylation-coupled Western blotting. Detection of deglycosylated Epo enables the examination of small changes in Epo production. Using this method, we investigated the effects of angiotensin II (ATII) on Epo production in the kidney. ATII stimulated the plasma Epo concentration; Epo, HIF2α, and PHD2 mRNA expression in nephron segments in the renal cortex and outer medulla; and Epo protein expression in the renal cortex. In situ hybridization and immunohistochemistry revealed that ATII stimulates Epo mRNA and protein expression not only in proximal tubules but also in collecting ducts, especially in intercalated cells. These data support the regulation of Epo production in the kidney by the renin–angiotensin–aldosterone system (RAS)

Targeted DNA methylation in pericentromeres with genome editing-based artificial DNA methyltransferase

<div><p>To study the impact of epigenetic changes on biological functions, the ability to manipulate the epigenetic status of certain genomic regions artificially could be an indispensable technology. “Epigenome editing” techniques have gradually emerged that apply TALE or CRISPR/Cas9 technologies with various effector domains isolated from epigenetic code writers or erasers such as DNA methyltransferase, 5-methylcytosine oxidase, and histone modification enzymes. Here we demonstrate that a TALE recognizing a major satellite, consisting of a repeated sequence in pericentromeres, could be fused with the bacterial CpG methyltransferase, SssI. ChIP-qPCR assays demonstrated that the fusion protein TALMaj-SssI preferentially bound to major chromosomal satellites in cultured cell lines. Then, TALMaj-SssI was expressed in fertilized mouse oocytes with hypomethylated major satellites (10–20% CpG islands). Bisulfite sequencing revealed that the DNA methylation status was increased specifically in major satellites (50–60%), but not in minor satellites or other repeat elements, such as Intracisternal A-particle (IAP) or long interspersed nuclear elements-1 (Line1) when the expression level of TALMaj-SssI is optimized in the cell. At a microscopic level, distal ends of chromosomes at the first mitotic stage were dramatically highlighted by the mCherry-tagged methyl CpG binding domain of human MBD1 (mCherry-MBD-NLS). Moreover, targeted DNA methylation to major satellites did not interfere with kinetochore function during early embryonic cleavages. Co-injection of dCas9 fused with SssI and guide RNA (gRNA) recognizing major satellite sequences enabled increment of the DNA methylation in the satellites, but a few off-target effects were also observed in minor satellites and retrotransposons. Although CRISPR can be applied instead of the TALE system, technical improvements to reduce off-target effects are required. We have demonstrated a new method of introducing DNA methylation without the need of other binding partners using the CpG methyltransferase, SssI.</p></div

Target dominant upregulation of DNA methylation by TALMaj-SssI expression in mouse embryos.

<p>(A) Bisulfite sequencing of TALMaj-SssI-expressing embryos. Methylation of major and minor satellite CpGs was evaluated in TALMaj-SssI embryos. TALMaj-SssI was expressed at 3 levels (low, middle, and high, from left to right) by injecting various concentrations (2, 10, and 50 ng/μL, respectively) of RNAs encoding TALMaj-SssI (WT or T313D) into fertilized embryos. Mock control indicates normal fertilized embryos without injection. Embryos were recovered 3 days after RNA injection. (B) DNA methylation of IAP and Line1 was evaluated in TALMaj-SssI-expressing embryos. Embryos expressing the middle level (10 ng/μL RNA injection) of TALMaj-SssI were evaluated. Mock control indicates normal fertilized embryos without injection. Data are expressed as the percentage of methylated CpG sites relative to all CpG sites. Asterisks indicate significant differences between the mock control and tested group by the Mann–Whitney <i>U</i> test.</p

Upregulation of major satellite DNA methylation does not hamper chromosome segregation in preimplantation embryos.

<p>(A) Chromosome segregation patterns observed in embryos expressing the TALMaj-SssI (WT) fusion protein. Snapshots of the 2-cell to 4-cell transition are indicated. Embryos were injected with RNAs encoding mCherry-MBD-NLS (5 ng/μL), H2B-EGFP (10 ng/μL) and TALMaj-SssI (WT or T313D; 10 ng/μL) and we observed EGFP fluorescence during the 1-cell to 8-cell stages by live-cell imaging. Embryos were classified into two groups: abnormal chromosome segregation (ACS) and normal chromosome segregation (NCS). Arrows indicate lagging chromosomes. Scale bar = 20 μm. (B) Frequency of ACS in embryos expressing TALMaj-SssI. These were classified as having 1–2 ACS, 2–4 ACS, or 4–8 ACS, based on the timing of ACS at the 1-cell to 2-cell, 2-cell to 4-cell and 4-cell to 8-cell stages, respectively. In all, 29 WT and 24 T313D embryos were assessed for ACS analysis. The ACS frequencies in these three groups were compared with embryos expressing the WT or T313D proteins by chi-squared test. No statistically significant difference was observed.</p