FOXO signaling pathway participates in oxidative stress-induced histone deacetylation.

High concentrations of antioxidants can exert pro-oxidative effects, elevate the level of intracellular reactive oxygen species (ROS), and cause oxidative stress in cells. We previously found that high concentrations of curcumin, a natural polyphenol antioxidant, elevated ROS levels and upregulated the expression of histone deacetylase 1 (HDAC1) in human gastric cancer cells (hGCCs); however, its potential mechanisms and subsequent functions have not been elucidated. In the present study, we treated hGCCs with high concentrations of curcumin, detected several indicators of oxidative stress, and investigated the mechanism of curcumin-treatment-mediated HDAC1 upregulation and its effect on histone acetylation. The results showed that curcumin treatment caused oxidative stress in hGCCs and upregulated HDAC1/2 expression via the forkhead box O (FOXO) signaling pathway, ultimately leading to the deacetylation of histones in hGCCs. Moreover, HDAC1/2 mediates the deacetylation of FOXOs and promotes their transcription activities, implying a positive feedback loop between FOXOs and HDAC1/2. These findings present a mechanism by which oxidative stress induces histone deacetylation in hGCCs.

Sncg, Mybpc1, and Parm1 Classify subpopulations of VIP-expressing interneurons in layers 2/3 of the somatosensory cortex.

Neocortical vasoactive intestinal polypeptide-expressing (VIP+) interneurons display highly diverse morpho-electrophysiological and molecular properties. To begin to understand the function of VIP+ interneurons in cortical circuits, they must be clearly and comprehensively classified into distinct subpopulations based on specific molecular markers. Here, we utilized patch-clamp RT-PCR (Patch-PCR) to simultaneously obtain the morpho-electric properties and mRNA profiles of 155 VIP+ interneurons in layers 2 and 3 (L2/3) of the mouse somatosensory cortex. Using an unsupervised clustering method, we identified 3 electrophysiological types (E-types) and 2 morphological types (M-types) of VIP+ interneurons. Joint clustering based on the combined electrophysiological and morphological features resulted in 3 morpho-electric types (ME-types). More importantly, we found these 3 ME-types expressed distinct marker genes: ~94% of Sncg+ cells were ME-type 1, 100% of Mybpc1+ cells were ME-type 2, and ~78% of Parm1+ were ME-type 3. By clarifying the properties of subpopulations of cortical L2/3 VIP+ interneurons, this study establishes a basis for future investigations aiming to elucidate their physiological roles.

α-Amylase inhibition of a certain dietary polyphenol is predominantly affected by the concentration of α-1, 4-glucosidic bonds in starchy and artificial substrates.

To elucidate why the inhibitory activity of one same polyphenol against α-amylase varies in different works. Seven starchy and three artificial substrates, and a polyphenolic competitive inhibitor, tannic acid (TA) were applied to study the enzyme inhibition in different digestion systems. The results showed that the IC50 values of TA were similar for all starches at the same starch concentration, although there existed difference in starch physiochemical properties, like branching degree, amylose chain distribution, viscosity, and digestion rate. However, the IC50 values significantly decreased with the substrate concentration decreasing, regardless of substrate types. Notably, TA had a similar competitive inhibition constant (Kic) for all the starches, despite the difference in substrate concentration, indicating that the fixed constant unconditionally describes the inhibitor-enzyme binding property. In the TA/amylase/starch system, the physical adsorption of TA with starch was much weaker than the specific binding of TA with α-amylase that was driven by hydrogen bondings and π-stackings. Therefore, it was the substrate, i.e., α-1,4-glucosidic bond concentration, rather than the existing matrix of the bonds, that predominantly affected the inhibitory activity of a polyphenol, because of the competitive action between α-1,4-glucosidic bond and the polyphenol regarding binding with the enzyme.

Number of galloyl moiety and intramolecular bonds in galloyl-based polyphenols affect their interaction with alpha-glucosidase.

The inhibition of α-glucosidase by nine galloyl-based polyphenols with free and unfree galloyl moieties (GMs) was studied. The results show that the inhibitory activity increased with the free GM number increasing. For the compounds with unfree GMs, ellagic acid and hexahydroxydiphenoyl group contributed to the enzyme inhibition. Free GMs could bind not only with the active site of α-glucosidase (competitive inhibition character), but also with the non-active sites (uncompetitive one); however, the former binding interaction was stronger than the latter one. All polyphenols that had inhibitory effects quenched α-glucosidase fluorescence in a static mode through forming a polyphenol-enzyme complex. The number of amino acid residues involved in polyphenol-enzyme binding interactions (hydrogen bonding and π-conjugations) increased with the inhibitory activity increasing. Additionally, two polyphenols with 5 free GMs showing certain hypoglycemic effects in maltose-loading test suggests that GM may be an advisable functional factor for alleviation of type II diabetes symptoms.

The Physical Adsorption of Gelatinized Starch with Tannic Acid Decreases the Inhibitory Activity of the Polyphenol against α-Amylase.

The effects of mixing orders of tannic acid (TA), starch, and α-amylase on the enzyme inhibition of TA were studied, including mixing TA with α-amylase before starch addition (order 1), mixing TA with pre-gelatinized starch before α-amylase addition (order 2) and co-gelatinizing TA with starch before α-amylase addition (order 3). It was found that the enzyme inhibition was always highest for order 1 because TA could bind with the enzyme active site thoroughly before digestion occurred. Both order 2 and 3 reduced α-amylase inhibition through decreasing binding of TA with the enzyme, which resulted from the non-covalent physical adsorption of TA with gelatinized starch. Interestingly, at low TA concentration, α-amylase inhibition for order 2 was higher than order 3, while at high TA concentration, the inhibition was shown with the opposite trend, which arose from the difference in the adsorption property between the pre-gelatinized and co-gelatinized starch at the corresponding TA concentrations. Moreover, both the crystalline structures and apparent morphology of starch were not significantly altered by TA addition for order 2 and 3. Conclusively, although a polyphenol has an acceptable inhibitory activity in vitro, the actual effect may not reach the expected one when taking processing procedures into account.

Number of galloyl moieties and molecular flexibility are both important in alpha-amylase inhibition by galloyl-based polyphenols.

The inhibition of porcine pancreatic α-amylase (PPA) by 9 galloyl-based polyphenols was evaluated via initial digestion velocity, IC50, inhibition kinetics, fluorescence quenching and molecular docking studies. The results show that the polyphenols with free galloyl moieties (GMs) exhibited a much higher inhibition effect than that with bound GMs. The inhibition was competitive mode and increased with an increase in the number of free GMs. Interestingly, all the constants that indicate the binding affinity of polyphenols to PPA, including the reciprocal of the competitive inhibition constant (1/Kic), fluorescence quenching constant (KFQ) and binding energy (Eb), increased with an increase in the number of free GMs. Besides, the respective order of 1/Kic, KFQ and Eb for the galloyl-based polyphenols was contrary to that of IC50, indicating that the polyphenol-PPA binding interactions resulted in enzyme inhibition. In addition, the binding interactions were suggested to result from the hydrogen bonding and π-π conjugation forces between the free GMs and amino acid residues at the active sites of PPA, whereas the chemical linkage of the GMs with additional polyphenol groups limited these interactions. Furthermore, the hypoglycemic effects of two polyphenols with 5 free GMs indicate that GMs may be considered a functional fragment involved in the alleviation of the symptoms of type II diabetes through α-amylase inhibition.

Physiological and genomic features of Paraoceanicella profunda gen. nov., sp. nov., a novel piezophile isolated from deep seawater of the Mariana Trench.

A novel piezophilic alphaproteobacterium, strain D4M1T , was isolated from deep seawater of the Mariana Trench. 16S rRNA gene analysis showed that strain D4M1T was most closely related to Oceanicella actignis PRQ-67T (94.2%), Oceanibium sediminis O448T (94.2%), and Thioclava electrotropha ElOx9T (94.1%). Phylogenetic analyses based on both 16S rRNA gene and genome sequences showed that strain D4M1T formed an independent monophyletic branch paralleled with the genus Oceanicella in the family Rhodobacteraceae. Cells were Gram-stain-negative, aerobic short rods, and grew optimally at 37°C, pH 6.5, and 3.0% (w/v) NaCl. Strain D4M1T was piezophilic with the optimum pressure of 10 MPa. The principal fatty acids were C18:1 ω7c/C18:1 ω6c and C16:0 , major respiratory quinone was ubiquinone-10, and predominant polar lipids were phosphatidylglycerol, phosphatidylethanolamine, and an unidentified aminophospholipid. The complete genome contained 5,468,583-bp with a G + C content of 70.2 mol% and contained 4,855 protein-coding genes and 78 RNA genes. Genomic analysis revealed abundant clues on bacterial high-pressure adaptation and piezophilic lifestyle. The combined evidence shows that strain D4M1T represents a novel species of a novel genus in the family Rhodobacteraceae, for which the name Paraoceanicella profunda gen. nov., sp. nov. is proposed (type strain D4M1T  = MCCC 1K03820T  = KCTC 72285T ).

Algoriphagus litoralis sp. nov., isolated from the junction between the ocean and a freshwater lake.

A Gram-stain negative, non-motile and short rod shaped bacterium, designated strain DSL-12T, was isolated from seawater of the East China Sea and characterised phylogenetically and phenotypically. Optimal growth was found to occur at 28 °C (range 4-40 °C), pH 7 (range 6-12) and with 3% (w/v) NaCl (range 0-8%). Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain DSL-12T is related to members of the genus Algoriphagus and shares high sequence similarities with Algoriphagus boritolerans DSM 17298T (97.6%) and Algoriphagus alkaliphilus DSM 22703T (97.6%). The 16S rRNA gene sequence identities between strain DSL-12T and other current members of the genus Algoriphagus were below 96.4%. The digital DNA-DNA hybridization values between strain DSL-12T and the type strains A. boritolerans DSM 17298T and A. alkaliphilus DSM 22703T were found to be 21.2 ± 2.4% and 20.2 ± 2.4%, respectively. The average nucleotide identity (ANI) values between strain DSL-12T and A. boritolerans DSM 17298T and A. alkaliphilus DSM 22703T were found to be 83.2% and 82.8%, respectively. The sole respiratory quinone was identified as MK-7. The major polar lipids were identified as phosphatidylethanolamine, diphosphatidylglycerol, one unidentified phospholipid and two unidentified lipids. The major fatty acids identified as were iso-C15:0, summed feature 8 (C18:1ω7c and/or C18:1ω6c), C16:0, summed feature 3 (C16:1ω7c and/or C16:1ω6c) and iso-C17:0. The G+C content of the genomic DNA was determined to be 43.3 mol%. The combined genotypic and phenotypic data indicate that strain DSL-12T represents a novel species of the genus Algoriphagus, for which the name Algoriphagus litoralis sp. nov. is proposed, with the type strain DSL-12T (= KCTC 62647T = MCCC 1K03536T).

Polyphasic taxonomic analysis of Parasedimentitalea marina gen. nov., sp. nov., a psychrotolerant bacterium isolated from deep sea water of the New Britain Trench.

A novel Rhodobacteraceae bacterium, strain W43T, was isolated from a deep-sea water sample from the New Britain Trench. Strain W43T exhibited the highest 16S rRNA gene sequence similarity of 96.5% to Sedimentitalea nanhaiensis DSM 24252T, Phaeobacter gallaeciensis DSM 26640T, Phaeobacter inhibens DSM 16374T, and Phaeobacter porticola P97T. Phylogenetic analysis of the 16S rRNA gene and phylogenomic analysis of the genome showed that strain W43T formed an independent monophyletic branch within the family Rhodobacteraceae. Strain W43T was Gram-stain-negative, rod-shaped, and grew optimally at 16-20°C, pH 6.5-7.0 and 2% (w/v) NaCl. The principal fatty acids were C18:1ω7c/C18:1ω6c, major respiratory quinone was ubiquinone-10 and predominant polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The 5 080 916 bp long genome, comprising a circular chromosome and four plasmids, exhibits a G + C content of 55.9 mol%. The combined phenotypic, chemotaxonomic and molecular data show that strain W43T represents a novel species of a novel genus, for which the name Parasedimentitalea marina gen. nov. sp. nov. is proposed (type strain W43T = MCCC 1K03532T = KCTC 62635T).

Marinobacter profundi sp. nov., a slightly halophilic bacterium isolated from a deep-sea sediment sample of the New Britain Trench.

A piezotolerant, cold-adapted, slightly halophilic bacterium, designated strain PWS21T, was isolated from a deep-sea sediment sample collected from the New Britain Trench. Cells were observed to be Gram-stain negative, rod-shaped, oxidase- and catalase-positive. Growth of the strain was observed at 4-45 °C (optimum 37 °C), at pH 5.0-9.0 (optimum 7.0) and in 0.5-20% (w/v) NaCl (optimum 3-4%). The optimum pressure for growth was 0.1 MPa (megapascal) with tolerance up to 70 MPa. 16S rRNA gene sequence analysis showed that strain PWS21T is closely related to Marinobacter guineae M3BT (98.4%) and Marinobacter lipolyticus SM19T (98.2%). Multilocus sequence analysis (MLSA) based on sequences of housekeeping genes gyrB, recA, atpD, rpoB and rpoD indicates that strain PWS21T represents a distinct evolutionary lineage within the genus Marinobacter. Furthermore, strain PWS21T showed low ANI and diDDH values to the closely related species. The principal fatty acids were identified as C12:0, C12:0 3-OH, C16:1ω9c, C16:0 and C18:1ω9c. Ubiquinone-9 was identified as the major respiratory quinone. The polar lipids were identified as phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), aminophospholipid (APL), two unidentified lipids and an unidentified phospholipid (PL). The G + C content of the genomic DNA was determined to be 60.3 mol%. On the basis of phenotypic, chemotaxonomic and molecular data, we conclude that strain PWS21T represents a novel species of the genus Marinobacter, for which the name Marinobacter profundi sp. nov. is proposed (type strain PWS21T = KCTC 52990T = MCCC 1K03345T).

PAX3-FOXO1 escapes miR-495 regulation during muscle differentiation.

Pax3 plays an essential role in myogenesis. Previously, we found a tumor-signature chimeric fusion RNA, PAX3-FOXO1 also present during muscle differentiation, raising the possibility of its physiological role. Here we demonstrated that the fusion is needed transiently for muscle lineage commitment. Interestingly, the fusion ortholog was not found in seven mouse muscle differentiation/regeneration systems, nor in other stem cell differentiation systems of another three mammal species. We noticed that Pax3 is expressed at a much lower level in human stem cells, and during muscle differentiation than in other mammals. Given the fact that the fusion and the parental Pax3 share common downstream targets, we reasoned that forming the fusion may be a mechanism for human cells to escape certain microRNA regulation on Pax3. By sequence comparison, we identified 16 candidate microRNAs that may specifically target the human PAX3 3'UTR. We used a luciferase reporter assay, examined the microRNAs expression, and conducted mutagenesis on the reporters, as well as a CRISPR/Cas9 mediated editing on the endogenous allele. Finally, we identified miR-495 as a microRNA that specifically targets human PAX3. Examining several other fusion RNAs revealed that the human-specificity is not limited to PAX3-FOXO1. Based on these observations, we conclude that PAX3-FOXO1 fusion RNA is absent in mouse, or other mammals we tested, the fusion RNA is a mechanism to escape microRNA, miR-495 regulation in humans, and that it is not the only human-specific fusion RNA.

Fear Erasure Facilitated by Immature Inhibitory Neuron Transplantation.

Transplantation of embryonic γ-aminobutyric acid (GABA)ergic neurons has been shown to modify disease phenotypes in rodent models of neurologic and psychiatric disorders. However, whether transplanted interneurons modulate fear memory remains largely unclear. Here, we report that transplantation of embryonic interneurons into the amygdala does not alter host fear memory formation. Yet approximately 2 weeks after transplantation, but not earlier or later, extinction training produces a marked reduction in spontaneous recovery and renewal of fear response. Further analyses reveal that transplanted interneurons robustly form functional synapses with neurons of the host amygdala and exhibit similar developmental maturation in electrophysiological properties as native amygdala interneurons. Importantly, transplanted immature interneurons reduce the expression of perineuronal nets, promote long-term synaptic plasticity, and modulate both excitatory and inhibitory synaptic transmissions of the host circuits. Our findings demonstrate that transplanted immature interneurons modify amygdala circuitry and suggest a previously unknown strategy for the prevention of extinction-resistant pathological fear.

Isolation, Culture, Differentiation, and Nuclear Reprogramming of Mongolian Sheep Fetal Bone Marrow-Derived Mesenchymal Stem Cells.

We have characterized the differentiation potentiality and the developmental potential of cloned embryos of fetal bone marrow mesenchymal stem cells (BMSCs) isolated from Mongolian sheep. BMSCs were harvested by centrifuging after the explants method and the mononuclear cells obtained were cultured. The isolated BMSCs were uniform, with a fibroblast-like spindle or stellate appearance, and we confirmed expression of OCT4, SOX2, and NANOG genes at passage 3 (P3) by RT-PCR. We measured the growth of the passage 1, 5, and 10 cultures and found exponential growth with a population doubling time of 29.7±0.05 h. We cultured the P3 BMSCs in vitro under inductive environments and were able to induce them to undergo neurogenesis and form cardiomyocytes and adipocytes. Donor cells at passages 3-4 were used for nuclear transfer (NT). We found the BMSCs could be expanded in vitro and used as nuclear donors for somatic cell nuclear transfer (SCNT). Thus, BMSCs are an attractive cell type for large-animal autologous studies and will be valuable material for somatic cell cloning and future transgenic research.

The development and expression of pluripotency genes in embryos derived from nuclear transfer and in vitro fertilization.

Somatic cell nuclear transfer can be used to produce embryonic stem (ES) cells, cloned animals, and can even increase the population size of endangered animals. However, the application of this technique is limited by the low developmental rate of cloned embryos, a situation that may result from abnormal expression of some zygotic genes. In this study, sheep-sheep intra-species cloned embryos, goat-sheep inter-species cloned embryos, or sheep in vitro fertilized embryos were constructed and cultured in vitro and the developmental ability and expression of three pluripotency genes, SSEA-1, Nanog and Oct4, were examined. The results showed firstly that the developmental ability of in vitro fertilized embryos was significantly higher than that of cloned embryos. In addition, the percentage of intra-species cloned embryos that developed to morula or blastocyst stages was also significantly higher than that of the inter-species cloned embryos. Secondly, all three types of embryos expressed SSEA-1 at the 8-cell and morula stages. At the 8-cell stage, a higher percentage of in vitro fertilized embryos expressed SSEA-1 than occurred for cloned embryos. However, at the morula stage, all detected embryos could express SSEA-1. Thirdly, the three types of embryos expressed Oct4 mRNA at the morula and blastocyst stages, and embryos at the blastocyst stage expressed Nanog mRNA. The rate of expression of Oct4 and Nanog mRNA at these developmental stages was higher in in vitro fertilized embryos than in cloned embryos. These results indicated that, during early development, the failure to reactivate some pluripotency genes maybe is a reason for the low cloning efficiency found with cloned embryos.