Elucidation of mechanism of Si-jun-zi decoction-induced reversal of spleen deficiency syndrome in rats by LC-QTOF/ MS metabolomics

Purpose: To explore the therapeutic effect and mechanism of action of Si–jun–zi decoction (SJZD) in rat spleen deficiency syndrome.Methods: Spleen deficiency syndrome was induced in a rat model with reserpine. Liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF/MS) and partial least squaresdiscriminate models (PLS-DA) were used to analyze the difference in global metabolite profile within all groups. Variable importance projection (VIP > 1) and Student’s t–test (p < 0.05) were used for biomarker selection.Results: Enzymatic results showed that the level of amylase (AMY), pepsin (LPS) and pepsase were significantly lower in the untreated rats than in normal control rats and SJZD group rats (P < 0.05). Histopathological analysis showed there were significant pathological changes in the untreated rats compared with the normal control group and SJZD group rats. PLS-DA score plots of the untreated group rats clustered separately from control rats, whereas SJZD group rats clustered closely to normal control rats. Twenty potential biomarkers, including phospholipid and fatty acids, were screened. The relative content of 20 metabolites showed significant differences in the untreated group compared with control and SJZD groups (p < 0.05). Six potentially perturbed metabolic pathways were found, which might be potential targets pathways for SJZD intervention in spleen deficiency syndrome.Conclusion: Metabolomics approach is an effective tool for understanding the  therapeutic mechanisms of SJZD.Keywords: Si–jun–zi decoction, Spleen deficiency syndrome, Metabolomics,  LC-Q-TOF/M

MicroRNAs control mRNA fate by compartmentalization based on 3 ' UTR length in male germ cells

Post-transcriptional regulation of gene expression can be achieved through the control of mRNA stability, cytoplasmic compartmentalization, 3' UTR length and translational efficacy. Spermiogenesis, a process through which haploid male germ cells differentiate into spermatozoa, represents an ideal model for studying post-transcriptional regulation in vivo because it involves a large number of transcripts that are physically sequestered in ribonucleoprotein particles (RNPs) and thus subjected to delayed translation. To explore how small RNAs regulate mRNA fate, we conducted RNA-Seq analyses to determine not only the levels of both mRNAs and small noncoding RNAs, but also their cytoplasmic compartmentalization during spermiogenesis. Result: Among all small noncoding RNAs studied, miRNAs displayed the most dynamic changes in both abundance and subcytoplasmic localization. mRNAs with shorter 3' UTRs became increasingly enriched in RNPs from pachytene spermatocytes to round spermatids, and the enrichment of shorter 3' UTR mRNAs in RNPs coincided with newly synthesized miRNAs that target these mRNAs at sites closer to the stop codon. In contrast, the translocation of longer 3' UTR mRNAs from RNPs to polysomes correlated with the production of new miRNAs that target these mRNAs at sites distal to the stop codon. Conclusions: miRNAs appear to control cytoplasmic compartmentalization of mRNAs based on 3' UTR length. Our data suggest that transcripts with longer 3' UTRs tend to contain distal miRNA binding sites and are thus targeted to polysomes for translation followed by degradation. In contrast, those with shorter 3' UTRs only possess proximal miRNA binding sites, which, therefore, are targeted into RNPs for enrichment and delayed translation

Bfd, a New Class of [2Fe-2S] Protein That Functions in Bacterial Iron Homeostasis, Requires a Structural Anion Binding Site

Copyright © 2018 American Chemical Society. Mobilization of iron from bacterioferritin (BfrB) requires specific interactions with a [2Fe-2S] ferredoxin (Bfd). Blocking the BfrB:Bfd interaction results in irreversible iron accumulation in BfrB and iron deficiency in the cytosol [Eshelman, K., et al. (2017) Metallomics 9, 646-659]. The only known Bfd structure, which was obtained in complex with BfrB (Protein Data Bank entry 4E6K), indicated a new fold and suggested that the stability of Bfd is aided by an anion binding site consisting of R26, R29, and K46. We investigated the Bfd fold using site-directed mutagenesis, X-ray crystallography, and biochemistry in solution. The X-ray structure, which is nearly identical to that of Bfd in the BfrB:Bfd complex, shows that the [2Fe-2S] cluster preorganizes residues at the BfrB:Bfd interface into a structure complementary to the Bfd binding site on BfrB. Studies in solution showed rapid loss of the [2Fe-2S] cluster at a low ionic strength but higher stability with an increasing ionic strength, thus supporting a structural anion binding site. Structures of the R26E and R26E/K46Y mutants are nearly identical to that of Bfd, except for a new network of hydrogen bonds stabilizing the region encompassing the former anion binding site. The stability of the R26E and R26E/K46Y mutants, which is weakly and completely independent of solution ionic strength, respectively, corroborates that Bfd requires an anion binding site. The mutations, which caused only small changes to the strength of the BfrB:Bfd interaction and mobilization of iron from BfrB, indicate that the anion binding site in Bfd serves primarily a structural role