Transcriptome Analysis Reveals the Putative Polyketide Synthase Gene Involved in Hispidin Biosynthesis in <i>Sanghuangporus sanghuang</i>

Hispidin is an important styrylpyrone produced by Sanghuangporus sanghuang. To analyze hispidin biosynthesis in S. sanghuang, the transcriptomes of hispidin-producing and non-producing S. sanghuang were determined by Illumina sequencing. Five PKSs were identified using genome annotation. Comparative analysis with the reference transcriptome showed that two PKSs (ShPKS3 and ShPKS4) had low expression levels in four types of media. The gene expression pattern of only ShPKS1 was consistent with the yield variation of hispidin. The combined analyses of gene expression with qPCR and hispidin detection by liquid chromatography-mass spectrometry coupled with ion-trap and time-of-flight technologies (LCMS-IT-TOF) showed that ShPKS1 was involved in hispidin biosynthesis in S. sanghuang. ShPKS1 is a partially reducing PKS gene with extra AMP and ACP domains before the KS domain. The domain architecture of ShPKS1 was AMP-ACP-KS-AT-DH-KR-ACP-ACP. Phylogenetic analysis shows that ShPKS1 and other PKS genes from Hymenochaetaceae form a unique monophyletic clade closely related to the clade containing Agaricales hispidin synthase. Taken together, our data indicate that ShPKS1 is a novel PKS of S. sanghuang involved in hispidin biosynthesis.</p

Novel TEMPO-PEG-RGDs Conjugates Remediate Tissue Damage Induced by Acute Limb Ischemia/Reperfusion

We have recently developed new Tempo-PEG-RGDs conjugates and have quantitatively examined their antithrombotic and antioxidant capabilities. These compounds were therapeutically beneficial when characterized in both in vitro platelet aggregation assays and a rat model of arterial thrombosis. Moreover, these compounds demonstrated significant protection from organ damage in a rat model of ischemia/reperfusion. Our data indicate that Tempo-PEG-RGDs represent a new class of adjuvants with therapeutic efficacy in acute and transient ischemic damage

Rifampicin-induced neuroprotection was GRP78-dependent.

<p>PC12 cells were transfected with GRP78-specific siRNAs or control siRNAs for 24 h. After that, cells were treated with or without 150 μM rifampicin for 2 h, followed by 1 μM rotenone for 24 h. (A) Western blot analysis verified the efficient gene silencing of GRP78. (B) After the above treatment, cell viability was measured and presented as the relative viability (% control). (C) Morphological evaluation of PC12 cells under the above-mentioned treatment by light microscopic observation and DAPI staining. The apoptotic cells were marked with arrows. Scale bar = 25 μm. (B–C) GRP78 gene silencing significantly exacerbated rotenone-triggered neuron injury, with or without the presence of rifampicin. Neither GRP78-specific nor control siRNAs decreased cell viability. Data present mean ± SEM of three independent experiments. *p<0.05.</p

2D-DIGE gel images of proteins isolated from rifampicin-treated PC12 cells.

<p>(A) Arrows indicate proteins that were differentially expressed in PC12 cells treated with or without rifampicin. (B) Representative peptide mass fingerprint spectra generated by MALDI-TOF-MS. The x-axis indicates the mass-to-charge ratio, m/z. The y-axis indicates the relative abundance. Peptide masses are labeled and the corresponding m/z is annotated.</p

No significant activation of the IREα-XBP1 or ATF6 pathway by rifampicin.

<p>PC12 cells were incubated with 150 μM rifampicin for indicated lengths of time, ranging from 3 to 24 h. Cells treated with 1 μM Tg served as positive controls. (A) Cell lysates were subjected to western blotting using p-IREα and β-actin antibodies. Rifampicin did not stimulate IREα phosphorylation. (B) Total RNA was subjected to RT-PCR to measure XBP1u/XBP1s expression. Rifampicin did not induce splicing of XBP1 mRNA significantly. (C) Cell lysates were analyzed by western blotting using the ATF6 antibody. Rifampicin did not activate ATF6 cleavage in PC12 cells. Data present mean ± SEM of three independent experiments, with four to six replicates each.</p

MSS1 gene knockdown reduced the expression of MSS1 at protein levels.

<p>In order to assess the efficacy of gene silencing, western blot analysis was performed after transfection with siRNAs targeting MSS1. The specificity of MSS1 gene silencing was determined by comparing with cells transfected with the scrambled RNA duplex. The BV2 cells were transfected with either MSS1-specfic or control siRNAs. At 24 h post-incubation, cell lysates were analyzed for the protein expression of MSS1 using western blot. Compared with the negative control group, the expression of MSS1 was significantly reduced by incubation with MSS1-targeted siRNAs. Data were obtained from three independent experiments with four to six replicates each. *<i>p</i><0.05 compared with the negative control group.</p

Rifampicin significantly suppressed the expression of MSS1 in LPS-stimulated BV2 microglia.

<p>Cells were treated with the indicated doses of rifampicin for 2 h prior to the addition of LPS (1000 ng/ml). At 24 h post-LPS incubation, cell lysates were analyzed for the protein production of MSS1 using western blot. Rifampicin significantly inhibited the LPS-induced MSS1 expression at protein levels. Data were obtained from three independent experiments with four to six replicates each. *<i>p</i><0.05 compared with untreated cells and cells treated with LPS in the absence of rifampicin.</p

Decreased iNOS expression and NO production by MSS1 gene silencing in LPS-activated microglia.

<p>The BV2 cells were transfected with either MSS1-specfic or control siRNA for 24 h, then cells were stimulated for 24 h with LPS (1000 ng/mL). At 24 h post-LPS incubation, cell lysates were analyzed for the protein production of iNOS using western blot. The Griess assay was performed to measure the production of the NO metabolite, nitrite. Transfection with MSS1-specific siRNA suppressed the LPS-induced iNOS expression at protein levels, along with the production of nitrites Data were obtained from three independent experiments with four to six replicates each. *<i>p</i><0.05 compared with the negative control group.</p

MSS1 gene silencing decreased IkBα protein degradation in LPS-activated microglia.

<p>The BV2 cells were transfected with either MSS1-specfic or control siRNAs for 24 h, then cells were stimulated with LPS (1000 ng/mL) for 30 min before cell lysates were analyzed for IkBα expression using western blot. IkBα protein degradation was significantly reduced after the addition of siRNAs targeting MSS1 in LPS-induced BV2 microglia. Data were obtained from three independent experiments with four to six replicates each. *p<0.05 compared with the negative control group.</p

Differential MSS1 protein expression identified by MALDI-TOF-MS.

<p>Differential MSS1 protein expression identified by MALDI-TOF-MS.</p