A predictive thermodynamic model for the bioreduction of acetophenone to phenethyl alcohol using resting cells of Saccharomyces cerevisiae.

Equilibrium conversions were observed in the range of 60.2-76.0% with different initial compositions of reaction media for the bioreduction of acetophenone using resting cells of Saccharomyces cerevisiae in aqueous solutions at 30 degrees C. The reduction of acetophenone in the cells under anaerobic conditions is considered to be coupled with the oxidation of ethanol to acetate in the cytoplasm. A biphasic thermodynamic model is proposed which includes a nonuniform distribution of reagents across the cell membrane, a transmembrane pH gradient, ideal and nonideal solution models, and a basic reaction stoichiometry (ACP + (1/2) EtOH + (1/2)H2O <--> PEA + (1/2)Ac- + (1/2)H+). The intracellular activity coefficients were based on the Lewis-Randall rule for acetophenone, phenethyl alcohol, and H2O and Henry's law for ethanol, acetate anion, and H+. The overall standard Gibbs free energy was estimated to be -0.11 kcal/mol at a pH 7, 25 degrees C, and 1 atm. The intracellular thermodynamic activity coefficients of acetophenone and phenethyl alcohol were predicted to be 471.2 and 866.4, respectively, using the measured initial distribution coefficients and calculated extracellular activity coefficients. The model reflected a zero Gibbs free energy change at calculated conversions within 4% of the measured equilibrium conversions. The analysis verified the effect of the concentration ratio of the substrate acetophenone to the co-substrate ethanol on the conversion efficiency and suggested that the intracellular pH and the pH gradient across the cell transmembrane significantly affect the predicted equilibrium conversion. The intracellular pH of resting, viable cells of Bakers' yeast at the bioconversion conditions was determined experimentally to be 5.77.     

The atlas of RNase H antisense oligonucleotide distribution and activity in the CNS of rodents and non-human primates following central administration

Antisense oligonucleotides (ASOs) have emerged as a new class of drugs to treat a wide range of diseases, including neurological indications. Spinraza, an ASO that modulates splicing of SMN2 RNA, has shown profound disease modifying effects in Spinal Muscular Atrophy (SMA) patients, energizing efforts to develop ASOs for other neurological diseases. While SMA specifically affects spinal motor neurons, other neurological diseases affect different central nervous system (CNS) regions, neuronal and non-neuronal cells. Therefore, it is important to characterize ASO distribution and activity in all major CNS structures and cell types to have a better understanding of which neurological diseases are amenable to ASO therapy. Here we present for the first time the atlas of ASO distribution and activity in the CNS of mice, rats, and non-human primates (NHP), species commonly used in preclinical therapeutic development. Following central administration of an ASO to rodents, we observe widespread distribution and target RNA reduction throughout the CNS in neurons, oligodendrocytes, astrocytes and microglia. This is also the case in NHP, despite a larger CNS volume and more complex neuroarchitecture. Our results demonstrate that ASO drugs are well suited for treating a wide range of neurological diseases for which no effective treatments are available.     

MicroRNA-205 affects mouse granulosa cell apoptosis and estradiol synthesis by targeting CREB1

MicroRNA-205 (miR-205) is involved in various physiological and pathological processes, but its biological function in follicular atresia remains unclear. In this study, we investigated miR-205 expression in mouse granulosa cells (mGCs) and analyzed its functions in primary mGCs by performing a series of in vitro experiments. Quantitative real-time polymerase chain reaction showed that miR-205 expression was significantly higher in early atretic follicles and progressively atretic follicles than in healthy follicles. miR-205 overexpression in mGCs significantly promoted apoptosis and caspase-3/9 activities, as well as inhibited estrogen (E2) release and cytochrome P450 family 19 subfamily A polypeptide 1 (CYP19A1, a key gene in E2 production) expression. Bioinformatics and luciferase reporter assays revealed that the gene encoding cyclic AMP response element (CRE)-binding protein 1 (CREB1) was a direct target of miR-205 in mGCs. CREB1 upregulation partially rescued the effects of miR-205 on apoptosis, caspase-3/9 activities, E2 production, and CYP19A1 expression on mGCs. These results indicate that miR-205 might play an important role in ovarian follicular development and provide new insights into follicular atresia     

Relationship of Nitrogen Deficiency-Induced Leaf Senescence with ROS Generation and ABA Concentration in Rice Flag Leaves

Nitrogen (N) deficiency is one of the critical environmental factors that induce leaf senescence, and its occurrence may cause the shorten leaf photosynthetic period and markedly lowered grain yield. However, the physiological metabolism underlying N deficiency-induced leaf senescence and its relationship with the abscisic acid (ABA) concentration and reactive oxygen species (ROS) burst in leaf tissues are not well understood. In this paper, the effect of N supply on several senescence-related physiological parameters and its relation to the temporal patterns of ABA concentration and ROS accumulation during leaf senescence were investigated using the premature senescence of flag leaf mutant rice (psf) and its wild type under three N treatments. The results showed that N deficiency hastened the initiation and progression of leaf senescence, and this occurrence was closely associated with the upregulated expression of 9-cis-epoxycarotenoiddioxygenase genes (NCEDs) and with the downregulated expression of two ABA 8′-hydroxylase isoform genes (ABA8ox2 and ABA8ox3) under LN treatment. Contrarily, HN supply delayed the initiation and progression of leaf senescence, concurrently with the suppressed ABA biosynthesis and relatively lower level of ABA concentration in leaf tissues. Exogenous ABA incubation enhanced ROS generation and MDA accumulation in a dose-dependent manner, but it decreased the activities of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in detached leaf. These results suggested that the participation of ABA in the regulation of ROS generation and N assimilating/remobilizing metabolism in rice leaves was strongly responsible for induction of leaf senescence by N deficiency.

     

Alterations and Mechanism of Gut Microbiota in Graves’ Disease and Hashimoto’s Thyroiditis

To explore the role of gut microbiota in Graves’ disease (GD) and Hashimoto’s thyroiditis (HT). Seventy fecal samples were collected, including 27 patients with GD, 27 with HT, and 16 samples from healthy volunteers. Chemiluminescence was used to detect thyroid function and autoantibodies (FT3, FT4, TSH, TRAb, TGAb, and TPOAb); thyroid ultrasound and 16S sequencing were used to analyze the bacteria in fecal samples; KEGG (Kyoto Encyclopedia of Genes and Genomes) and COG (Clusters of Orthologous Groups) were used to analyze the functional prediction and pathogenesis. The overall structure of gut microbiota in the GD and HT groups was significantly different from the healthy control group. Proteobacteria and Actinobacteria contents were the highest in the HT group. Compared to the control group, the GD and HT groups had a higher abundance of Erysipelotrichia, Cyanobacteria, and Ruminococcus_2 and lower levels of Bacillaceae and Megamonas. Further analysis of KEGG found that the “ABC transporter” metabolic pathway was highly correlated with the occurrence of GD and HT. COG analysis showed that the GD and HT groups were enriched in carbohydrate transport and metabolism compared to the healthy control group but not in amino acid transport and metabolism. Our data suggested that Bacillus, Blautia, and Ornithinimicrobium could be used as potential markers to distinguish GD and HT from the healthy population and that “ABC transporter” metabolic pathway may be involved in the pathogenesis of GD and HT.     

Sequence requirements for the termination of rolling-circle replication of plasmid pT181

Most small multicopy plasmids of Gram-positive bacteria and many in Gram-negative bacteria replicate by a rolling-circle (RC) mechanism. The replication initiator proteins encoded by the RC plasmids and single-stranded bacteriophages of Escherichia coli have origin-specific nicking-closing activities that are required for the initiation and termination of RC replication. We have investigated the sequence requirements for termination of RC replication of plasmid pT181. The initiator nick site is located in the loop of a hairpin region (IRII) within the pT181 origin of replication. By mutational analysis, we have found that several nucleotides within the stem of IRII which are critical for the initiation activity are dispensable for termination of replication. We also demonstrate that nucleotides in the right arm of IRII, but not the left arm, are absolutely required for termination of RC replication. We have also identified specific nucleotides in IRII that are critical for its termination activity. The sequence of the right arm of the hairpin must be located downstream of the initiator nick site for termination, suggesting that termination requires a specific orientation of the initiator protein at the origin.     

Network-assisted analysis to prioritize GWAS results: principles, methods and perspectives

Genome-wide association studies (GWAS) have rapidly become a powerful tool in genetic studies of complex diseases and traits. Traditionally, single marker-based tests have been used prevalently in GWAS and have uncovered tens of thousands of disease-associated SNPs. Network-assisted analysis (NAA) of GWAS data is an emerging area in which network-related approaches are developed and utilized to perform advanced analyses of GWAS data in order to study various human diseases or traits. Progress has been made in both methodology development and applications of NAA in GWAS data, and it has already been demonstrated that NAA results may enhance our interpretation and prioritization of candidate genes and markers. Inspired by the strong interest in and high demand for advanced GWAS data analysis, in this review article, we discuss the methodologies and strategies that have been reported for the NAA of GWAS data. Many NAA approaches search for subnetworks and assess the combined effects of multiple genes participating in the resultant subnetworks through a gene set analysis. With no restriction to pre-defined canonical pathways, NAA has the advantage of defining subnetworks with the guidance of the GWAS data under investigation. In addition, some NAA methods prioritize genes from GWAS data based on their interconnections in the reference network. Here, we summarize NAA applications to various diseases and discuss the available options and potential caveats related to their practical usage. Additionally, we provide perspectives regarding this rapidly growing research area.