Salt-induced phosphoproteomic changes in the subfornical organ in rats with chronic kidney disease

Subfornical organ (SFO) is vital in chronic kidney disease (CKD) progression caused by high salt levels. The current study investigated the effects of high salt on phosphoproteomic changes in SFO in CKD rats. 5/6 nephrectomized rats were fed a normal-salt diet (0.4%) (NC group) or a high-salt diet (4%) (HC group) for three weeks, while sham-operated rats were fed a normal-salt diet (0.4%) (NS group). For phosphoproteomic analysis of SFO in different groups, TiO2 enrichment, isobaric tags for relative and absolute quantification (iTRAQ) labeling, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used. There were 6808 distinct phosphopeptides found, which corresponded to 2661 phosphoproteins. NC group had 168 upregulated and 250 downregulated phosphopeptides compared to NS group. Comparison to NC group, HC group had 154 upregulated and 124 downregulated phosphopeptides. Growth associated protein 43 (GAP43) and heat shock protein 27 (Hsp27) were significantly upregulated phosphoproteins and may protect against high-salt damage. Differential phosphoproteins with tight functional connection were synapse proteins and microtubule-associated proteins, implying that high-salt diet disrupted brain’s structure and function. Furthermore, differential phosphoproteins in HC/NC comparison group were annotated to participate in GABAergic synapse signaling pathway and aldosterone synthesis and secretion, which attenuated inhibitory neurotransmitter effects and increased sympathetic nerve activity (SNA). This large scale phosphoproteomic profiling of SFO sheds light on how salt aggravates CKD via the central nervous system.</p

A Microporous Metal–Organic Framework with Lewis Basic Nitrogen Sites for High C₂H₂ Storage and Significantly Enhanced C₂H₂/CO₂ Separation at Ambient Conditions

A novel metal–organic framework (MOF), [Cu₂L­(H₂O)₂]·7DMF·4H₂O [<b>ZJU-40</b>; H₄L = 5,5′-(pyrazine-2,5-diyl)­diisophthalic acid], with Lewis basic nitrogen sites has been constructed and structurally characterized. Owing to the combined features of high porosity, moderate pore sizes, and immobilized Lewis basic nitrogen sites, the activated <b>ZJU-40a</b> exhibits the second-highest gravimetric C₂H₂ uptake of 216 cm³ g^–1 (at 298 K and 1 bar) among all of the reported MOFs so far. This value is not only much higher than that of the isoreticular <b>NOTT-101a</b> (184 cm³ g^–1), but also superior to those of two very promising MOFs, known as <b>HKUST-1</b> (201 cm³ g^–1) and <b>Co-MOF-74</b> (197 cm³ g^–1). Interestingly, the immobilized nitrogen sites in <b>ZJU-40a</b> have nearly no effect on the CO₂ uptake, so <b>ZJU-40a</b> adsorbs a similar amount of CO₂ (87 cm³ g^–1) compared with <b>NOTT-101a</b> (84 cm³ g^–1) at 298 K and 1 bar. As a result, <b>ZJU-40a</b> shows significantly enhanced adsorption selectivity for C₂H₂/CO₂ separation (17–11.5) at ambient temperature compared to that of <b>NOTT-101a</b> (8–9), leading to a superior MOF material for highly selective C₂H₂/CO₂ separation

A Microporous Metal–Organic Framework with Lewis Basic Nitrogen Sites for High C₂H₂ Storage and Significantly Enhanced C₂H₂/CO₂ Separation at Ambient Conditions

A novel metal–organic framework (MOF), [Cu₂L­(H₂O)₂]·7DMF·4H₂O [<b>ZJU-40</b>; H₄L = 5,5′-(pyrazine-2,5-diyl)­diisophthalic acid], with Lewis basic nitrogen sites has been constructed and structurally characterized. Owing to the combined features of high porosity, moderate pore sizes, and immobilized Lewis basic nitrogen sites, the activated <b>ZJU-40a</b> exhibits the second-highest gravimetric C₂H₂ uptake of 216 cm³ g^–1 (at 298 K and 1 bar) among all of the reported MOFs so far. This value is not only much higher than that of the isoreticular <b>NOTT-101a</b> (184 cm³ g^–1), but also superior to those of two very promising MOFs, known as <b>HKUST-1</b> (201 cm³ g^–1) and <b>Co-MOF-74</b> (197 cm³ g^–1). Interestingly, the immobilized nitrogen sites in <b>ZJU-40a</b> have nearly no effect on the CO₂ uptake, so <b>ZJU-40a</b> adsorbs a similar amount of CO₂ (87 cm³ g^–1) compared with <b>NOTT-101a</b> (84 cm³ g^–1) at 298 K and 1 bar. As a result, <b>ZJU-40a</b> shows significantly enhanced adsorption selectivity for C₂H₂/CO₂ separation (17–11.5) at ambient temperature compared to that of <b>NOTT-101a</b> (8–9), leading to a superior MOF material for highly selective C₂H₂/CO₂ separation

Androgen-Responsive MicroRNAs in Mouse Sertoli Cells

<div><p>Although decades of research have established that androgen is essential for spermatogenesis, androgen's mechanism of action remains elusive. This is in part because only a few androgen-responsive genes have been definitively identified in the testis. Here, we propose that microRNAs – small, non-coding RNAs – are one class of androgen-regulated <em>trans</em>-acting factors in the testis. Specifically, by using androgen suppression and androgen replacement in mice, we show that androgen regulates the expression of several microRNAs in Sertoli cells. Our results reveal that several of these microRNAs are preferentially expressed in the testis and regulate genes that are highly expressed in Sertoli cells. Because androgen receptor-mediated signaling is essential for the pre- and post-meiotic germ cell development, we propose that androgen controls these events by regulating Sertoli/germ cell-specific gene expression in a microRNA-dependent manner.</p> </div

Expression pattern of testosterone-responsive miRNAs.

<p>Real-time RT-PCR analysis of selected miRNA expression in total cellular RNA prepared from the adult mouse tissues. All values are normalized against RNU19 levels. Bar graphs represent the mean fold increase ± SEM of miRNA expression over background for at least two RT reactions assayed in duplicate from three separate mice. Several miRNAs negatively regulated by the androgen were specifically expressed in the testis.</p

Androgen-dependent expression of FoxD1 in Sertoli cells.

<p>(<b>A</b>) Immunohistochemical analysis on testis sections from Sham, flutamide-acyline (Flut+Acy) and flutamide-acyline testosterone-replacement (Flut+Acy+T) mice, using antibodies against Foxd1 (1∶50) and Sox9 (1∶500). Insets show Sertoli cell-specific expression of Foxd1 in magnified testicular section from Sham, Flut+Acy, and Flut+Acy+T mice. (<b>B</b>) Real-time PCR analysis of <i>Foxd1</i> transcript levels in total cellular RNA isolated from the purified Sertoli cells from Sham, Flut+Acy, and Flut+Acy+T mice testes. All values are normalized against RNU19 levels. (<b>C</b>) Western blot analysis on total testicular lysates from Sham, Flut+Acy, and Flut+Acy+T mice by using anti-Foxd1 antibody (1∶1000). Tubulin was used as a loading control. Values below the gel were quantified using the Image J software. Foxd1 protein level for the control was set to 1. Arrowheads indicate Sertoli cells. All images were taken at magnification of 600×.</p

Androgen-dependent expression of Dsc1 in Sertoli cells.

<p>(<b>A</b>) Immunohistochemical analysis on testis sections from Sham, flutamide-acyline (Flut+Acy) and flutamide-acyline testosterone-replacement (Flut+Acy+T) mice, using antibodies against Dsc1 (1∶50) and Sox9 (1∶500). Insets show Sertoli cell-specific expression of Dsc1 in magnified testicular section from Sham, Flut+Acy, and Flut+Acy+T mice. (<b>B</b>) Real-time PCR analysis of <i>Dsc1</i> transcript levels in total cellular RNA isolated from the purified Sertoli cells from Sham, Flut+Acy, and Flut+Acy+T mice testes. All values are normalized against RNU19 levels. (<b>C</b>) Western blot analysis on total testicular lysates from Sham, Flut+Acy, and Flut+Acy+T mice by using anti-Dsc1 antibody (1∶250). Tubulin was used as a loading control. Values below the gel were quantified using the Image J software. Dsc1 protein level for the control was set to 1. All images were taken at magnification of 600×.</p

MiR-471 regulates expression of Foxs1 in Sertoli cells.

<p>(<b>A</b>) Putative miR-471 binding sequence in the <i>Foxd1</i> 3′ UTR. (<b>B</b>) We co-transfected 15P1 Sertoli cells with <i>Renilla</i> luciferase expression construct pRL-CMV and firefly luciferase construct containing pMIR-<i>Foxd1</i> 3′ UTR in the absence and presence of miR-471 mimic. We normalized firefly luciferase activity of each sample to <i>Renilla</i> luciferase activity. Graphs show mean ± SEM of three independent experiments (performed in duplicate for each experiment). * <i>p</i><0.01; *** <i>p</i><0.001. (<b>C</b>) Real-time RT-PCR analysis of miR-471-overexpressing cells by using <i>Foxd1</i>-specific primers. (<b>D</b>) Western blot analysis of 15P1 cells transfected with miR-471 mimic by using anti-Foxd1 antibody (1∶1000). Tubulin was used as a loading control. Gel photographs represent three independent experiments. Values below the gel were quantified using Image J software (<a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a>). Foxd1 protein level for the control was set to 1.</p

Dsc1 is a bona fide target of miR-471.

<p>(<b>A</b>) Putative miR-471 binding sequence in the <i>Dsc1</i> 3′ UTR. (<b>B</b>) We co-transfected 15P1 Sertoli cells with <i>Renilla</i> luciferase expression construct pRL-CMV and firefly luciferase construct containing pMIR-<i>Dsc1</i> 3′ UTR in the absence and presence of miR-471 mimic. We normalized firefly luciferase activity of each sample to <i>Renilla</i> luciferase activity. Graphs show mean ± SEM of three independent experiments (performed in duplicate for each experiment). * <i>p</i><0.01; *** <i>p</i><0.001. (<b>C</b>) Real-time RT-PCR analysis of miR-471-overexpressing cells by using <i>Dsc1</i>-specific primers. (<b>D</b>) Western blot analysis of 15P1 cells transfected with miR-471 mimic by using anti-Dsc1 antibody (1∶250). Actin was used as a loading control. Gel photographs represent three independent experiments. Values below the gel were quantified using the Image J software. Dsc1 protein level for the control was set to 1.</p