Lysine Succinylation of VBS Contributes to Sclerotia Development and Aflatoxin Biosynthesis in Aspergillus flavus

Aspergillus flavus is a common saprophytic and pathogenic fungus, and its secondary metabolic pathways are one of the most highly characterized owing to its aflatoxin (AF) metabolite affecting global economic crops and human health. Different natural environments can cause significant variations in AF synthesis. Succinylation was recently identified as one of the most critical regulatory post-translational modifications affecting metabolic pathways. It is primarily reported in human cells and bacteria with few studies on fungi. Proteomic quantification of lysine succinylation (Ksuc) exploring its potential involvement in secondary metabolism regulation (including AF production) has not been performed under natural conditions in A. flavus. In this study, a quantification method was performed based on tandem mass tag labeling and antibody-based affinity enrichment of succinylated peptides via high accuracy nano-liquid chromatography with tandem mass spectrometry to explore the succinylation mechanism affecting the pathogenicity of naturally isolated A. flavus strains with varying toxin production. Altogether, 1240 Ksuc sites in 768 proteins were identified with 1103 sites in 685 proteins quantified. Comparing succinylated protein levels between high and low AF-producing A. flavus strains, bioinformatics analysis indicated that most succinylated proteins located in the AF biosynthetic pathway were downregulated, which directly affected AF synthesis. Versicolorin B synthase is a key catalytic enzyme for heterochrome B synthesis during AF synthesis. Site-directed mutagenesis and biochemical studies revealed that versicolorin B synthase succinylation is an important regulatory mechanism affecting sclerotia development and AF biosynthesis in A. flavus. In summary, our quantitative study of the lysine succinylome in high/low AF-producing strains revealed the role of Ksuc in regulating AF biosynthesis. We revealed novel insights into the metabolism of AF biosynthesis using naturally isolated A. flavus strains and identified a rich source of metabolism-related enzymes regulated by succinylation.     

Combination of cyclohexane and piperazine based κ-opioid receptor agonists: Synthesis and pharmacological evaluation of trans,trans-configured perhydroquinoxalines

Desymmetrization of the pseudochiral (2r)-configured cyclohexane-1,2,3-triamines 8 with dimethyl oxalate led to racemic aminoquinoxaline-2,3-diones 9. Selective introduction of the κ pharmacophoric structural elements pyrrolidine and 3,4-dichlorophenylacetamide with a two-carbon distance afforded conformationally restricted κ agonists 13–15 based on the quinoxaline ring system. In competitive radioligand receptor binding studies the benzylamine 13b, the secondary amine 14b, and the carbamate 15 displayed high κ receptor affinity. The K_i value of the lead compound derived methoxycarbonyl derivative 15 is 9.7 nM. However, the κ affinity of 15 is exceeded by 13b and 14b with a basic functional group instead of the methoxycarbonyl group in 1-position of the quinoxaline system. The chlorine atoms of the dichlorophenylacetyl residue are essential, since the corresponding phenylacetyl analogs show considerably reduced κ affinity. The potent κ ligands 13b, 14b and 15 are selective over the related μ- and δ-opioid receptors, σ₁, σ₂ and NMDA receptors. In the [³⁵S]GTPγS-binding assay 13b behaved as partial agonist with lower activity than U-69,593.     

Ascorbic acid prolongs the viability and stability of isolated perfused lungs: A mechanistic study using 31P and hyperpolarized 13C nuclear magnetic resonance

Ex vivo lung perfusion (EVLP) has recently shown promise as a means of more accurately gauging the health of lung grafts and improving graft performance post-transplant. However, reperfusion of ischemic lung promotes the depletion of high-energy compounds and a progressive loss of normal mitochondrial function, and it remains unclear how and to what extent the EVLP approach contributes to this metabolic decline. Although ascorbate has been used to mitigate the effects of ischemia–reperfusion injury, the nature of its effects during EVLP are also not clear. To address these uncertainties, this study monitored the energy status of lungs during EVLP and after the administration of ascorbate using ³¹P and hyperpolarized ¹³C NMR (nuclear magnetic resonance). Our experiments demonstrated that the oxidative phosphorylation capacity and pyruvate dehydrogenase flux of lungs decline during ex vivo perfusion. The addition of ascorbate to the perfusate prolonged lung viability by 80% and increased the hyperpolarized ¹³C bicarbonate signal by a factor of 2.7. The effect of ascorbate is apparently due not to its antioxidant quality but rather to its ability to energize cellular respiration given that it increased the lung’s energy charge significantly, whereas other antioxidants (glutathione and α-lipoic acid) did not alter energy metabolism. During ascorbate administration, inhibition of mitochondrial complex I with rotenone depressed energy charge and shifted the metabolic state of the lung toward glycolysis; reenergizing the electron transport chain with TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) recovered metabolic activity. This indicates that ascorbate slows the decline of the ex vivo perfused lung’s mitochondrial activity through an independent interaction with the electron transport chain complexes.     

Expression patterns and association analysis of the porcine DHX58 gene

RIG-1 signalling is responsible for the detection of cytoplasmic viral RNA molecules. DEXH (Asp-Glu-X-His) box polypeptide 58 (encoded by DHX58) is a negative regulator of the RIG-1 signalling pathway. In human, the DHX58 gene can be upregulated and can inhibit the RIG-1 signalling pathway during viral infection. In this study, porcine DHX58 gene expression patterns were studied. According to our results, the porcine DHX58 gene was upregulated not only by the stimulation of Poly I:C but also by the stimulation of 1ipopolysaccharides (LPS). One polymorphism (g.4919G>C), detected in the ninth intron, was significantly associated with some blood parameters including the red cell distribution width of 1-day-old pigs and white blood cell counts, lymphocyte absolute counts, and platelet distribution width of 17-day-old pigs (P < 0.05). Moreover, the individuals with the genotype GG have a significantly higher mean white blood cell count than individuals with genotype CC or GC (P < 0.05). Our study indicates that DHX58 is an important gene that is associated with the immune response in swine.     

Some New Aspects of 2 × 2 Table Measures and Their Applications

The paper deals with the analysis of 2 × 2 table measures and describes several new possibilities for applying them. An estimate is given for HELLMICH's asymmetric K-measure and a possible extension of the contingency concept in a special 2×2 case is presented. The concept introduced and the recommended computer application of the 2×2 table measures is supported by new results obtained for the relationship between jaw joint pains and noises examined earlier by F. SCHMID and C. ZSCHEGE. With a larger number of observations the opportunity arises for a deeper analysis of this dental problem using path analysis. The plan of evaluation and the conncetion between the variables examined is illustrated by a path-diagram. The association tables determined on the basis of the (C) values introduced during the examination of jaw joint disturbances using association analysis draw attention to the special role of the left side of mastication. The paper raises ideas for further generalisations and indicates where further research is needed.     

CLIX: a search algorithm for finding novel ligands capable of binding proteins of known three-dimensional structure.

A computer algorithm, CLIX, capable of searching a crystallographic data-base of small molecules for candidates which have both steric and chemical likelihood of binding a protein of known three-dimensional structure is presented. The algorithm is a significant advance over previous strategies which consider solely steric or chemical requirements for binding. The algorithm is shown to be capable of predicting the correct binding geometry of sialic acid to a mutant influenza-virus hemagglutinin and of proposing a number of potential new ligands to this protein.