The role of N6‐methyladenosine modification on diapause in silkworm (Bombyx mori) strains that exhibit different voltinism

N6‐methyladenosine (m⁶A) plays a key role in regulating gene expression in myriad organisms. Diapause is an important plastic phenotype that allows insects to survive under specific environmental conditions. However, the diapause molecular mechanism remains unknown. In this study, we analyzed the phylogenetics of genes related to the m⁶A modification complex in the silkworm (Bombyx mori) based on identified sequences from other organisms. We detected the expression of these genes during different developmental phases from four strains with different voltinism. We also determined total m⁶A content in cells treated with different diapause hormone concentrations or eggs exposed to hydrochloric acid. Our data revealed that m⁶A‐modification‐related gene expression and m⁶A content were greater in diapause‐destinated compared to nondiapause‐destined strains. Our findings suggest that m⁶A modification may provide significant epigenetic regulation of diapause‐related genes in the silkworm.     

Control of box C/D snoRNP assembly by N6‐methylation of adenine

N⁶‐methyladenine is the most widespread mRNA modification. A subset of human box C/D snoRNA species have target GAC sequences that lead to formation of N⁶‐methyladenine at a key trans Hoogsteen‐sugar A·G base pair, of which half are methylated in vivo. The GAC target is conserved only in those that are methylated. Methylation prevents binding of the 15.5‐kDa protein and the induced folding of the RNA. Thus, the assembly of the box C/D snoRNP could in principle be regulated by RNA methylation at its critical first stage. Crystallography reveals that N⁶‐methylation of adenine prevents the formation of trans Hoogsteen‐sugar A·G base pairs, explaining why the box C/D RNA cannot adopt its kinked conformation. More generally, our data indicate that sheared A·G base pairs (but not Watson–Crick base pairs) are more susceptible to disruption by N⁶mA methylation and are therefore possible regulatory sites. The human signal recognition particle RNA and many related Alu retrotransposon RNA species are also methylated at N6 of an adenine that forms a sheared base pair with guanine and mediates a key tertiary interaction.     

Human METTL16 is a N6‐methyladenosine (m6A) methyltransferase that targets pre‐mRNAs and various non‐coding RNAs

N⁶‐methyladenosine (m⁶A) is a highly dynamic RNA modification that has recently emerged as a key regulator of gene expression. While many m⁶A modifications are installed by the METTL3–METTL14 complex, others appear to be introduced independently, implying that additional human m⁶A methyltransferases remain to be identified. Using crosslinking and analysis of cDNA (CRAC), we reveal that the putative human m⁶A “writer” protein METTL16 binds to the U6 snRNA and other ncRNAs as well as numerous lncRNAs and pre‐mRNAs. We demonstrate that METTL16 is responsible for N⁶‐methylation of A43 of the U6 snRNA and identify the early U6 biogenesis factors La, LARP7 and the methylphosphate capping enzyme MEPCE as METTL16 interaction partners. Interestingly, A43 lies within an essential ACAGAGA box of U6 that base pairs with 5′ splice sites of pre‐mRNAs during splicing, suggesting that METTL16‐mediated modification of this site plays an important role in splicing regulation. The identification of METTL16 as an active m⁶A methyltransferase in human cells expands our understanding of the mechanisms by which the m⁶A landscape is installed on cellular RNAs.     

Analysis of phenolic compounds in health care products by low‐pressure liquid‐chromatography with monolithic column and chemiluminescent detection

This paper presents a new application for monolithic columns with low‐pressure chromatographic separation using an flow injection analysis configuration with chemiluminescent detection for the determination of a mixture of phenolic compounds: phloroglucinol, 2,4‐dihydroxybenzoic acid, salicylic acid, methyl paraben and n‐propyl gallate. The procedure consists of the separation of these compounds on a reverse‐phase ultra‐short monolithic column with pH 3.0 acetate buffer and 5% acetonitrile as carrier phase. The detection is based on a chemiluminescence measurement coming from Ce(IV)–Rhodamine 6G chemistry with the incorporation of two different chemiluminescent chemical conditions in the chromatographic setup in order to enhance the sensitivity for the different phenolic compounds. All separation and detection variables were optimized to propose a determination method. The analysis is performed in 280?s, with the sampling frequency being some 13 h^?1. The calibration function is a double reciprocal function obtaining good results within two orders of magnitude. The limits of detection were 8.8 × 10 ^?8 m (phloroglucinol), 2.7 × 10 ^?8 m (2,4‐dihydroxybenzoic acid); 2.3 × 10 ^?8 m (salicylic acid); 5.2 × 10 ^?8 m (methyl paraben) and 4.1 × 10 ^?6 m (n‐propyl gallate), and the relative standard deviations at a medium level of the linear range were 4.4% (phloroglucinol), 2.8% (2,4‐dihydroxybenzoic acid), 5.2% (salicylic acid), 3.6% (methyl paraben) and 6.8% (n‐propyl gallate). The method was applied and validated satisfactorily for the determination of these compounds in healthcare products, comparing the results against an HPLC reference method. Copyright © 2009 John Wiley & Sons, Ltd.     

Osteoclastogenesis Modulatory Steroids from the South China Sea Gorgonian Coral Iciligorgia sp.

A new 9,11‐secosteroid, (22R)‐22‐acetoxy‐3β,6α,11‐trihydroxy‐9,11‐seco‐5α‐cholest‐7‐en‐9‐one, along with twelve known analogues were isolated from the South China Sea gorgonian coral Iciligorgia sp. Their structures were determined by the spectroscopic analysis and comparison with reported data. In an in vitro test of osteoclastic differentiation, three compounds exhibited significant down‐regulating effect whereas two compounds showed significant up‐regulating effect at 0.5 and 1.0 μm . This is the first report of the chemical investigation of the gorgonian Iciligorgia sp. The acetoxy substitution at C‐22 seems to play an important role in the regulating activity.     

RNA N6-methyladenosine modification suppresses replication of rice black streaked dwarf virus and is associated with virus persistence in its insect vector

N⁶ methylation of adenosine (m⁶A) was recently discovered to play a role in regulating the life cycle of various viruses by modifying viral and host RNAs. However, different studies on m⁶A effects on the same or different viruses have revealed contradictory roles for m⁶A in the viral life cycle. In this study, we sought to define the role of m⁶A on infection by rice black streaked dwarf virus (RBSDV), a double-stranded RNA virus, of its vector small brown planthopper (SBPH). Infection by RBSDV decreased the level of m⁶A in midgut cells of SBPHs. We then cloned two genes (LsMETTL3 and LsMETTL14) that encode m⁶A RNA methyltransferase in SBPHs. After interference with expression of the two genes, the titre of RBSDV in the midgut cells of SBPHs increased significantly, suggesting that m⁶A levels were negatively correlated with virus replication. More importantly, our results revealed that m⁶A modification might be the epigenetic mechanism that regulates RBSDV replication in its insect vector and maintains a certain virus threshold required for persistent transmission.     

Differential targeting of Tetrahymena ORC to ribosomal DNA and non‐rDNA replication origins

The Tetrahymena thermophila origin recognition complex (ORC) contains an integral RNA subunit, 26T RNA, which confers specificity to the amplified ribosomal DNA (rDNA) origin by base pairing with an essential cis‐acting replication determinant—the type I element. Using a plasmid maintenance assay, we identified a 6.7 kb non‐rDNA fragment containing two closely associated replicators, ARS1‐A (0.8 kb) and ARS1‐B (1.2 kb). Both replicators lack type I elements and hence complementarity to 26T RNA, suggesting that ORC is recruited to these sites by an RNA‐independent mechanism. Consistent with this prediction, although ORC associated exclusively with origin sequences in the 21 kb rDNA minichromosome, the interaction between ORC and the non‐rDNA ARS1 chromosome changed across the cell cycle. In G₂ phase, ORC bound to all tested sequences in a 60 kb interval spanning ARS1‐A/B. Remarkably, ORC and Mcm6 associated with just the ARS1‐A replicator in G₁ phase when pre‐replicative complexes assemble. We propose that ORC is stochastically deposited onto newly replicated non‐rDNA chromosomes and subsequently targeted to preferred initiation sites prior to the next S phase.     

Enantiomeric separation of triazole fungicides with 3-μm and 5-μml particle chiral columns by reverse-phase high-performance liquid chromatography

This study used chiral columns packed with 3‐μm and 5‐μm particles to comparatively separate enantiomers of 9 triazole fungicides, and Lux Cellulose‐1 columns with chiral stationary phase of cellulose‐tris‐(3,5‐dimethylphenylcarbamate) were used on reverse‐phase high‐performance liquid chromatography with flow rates of 0.3 and 1.0 mL min⁻¹ for 3‐μm and 5‐μm columns, respectively. The (+)‐enantiomers of hexaconazole ( 1 ) , tetraconazole ( 4 ) , myclobutanil ( 7 ) , fenbuconazole ( 8 ) and the (−)‐enantiomers of flutriafol ( 2 ) diniconazole ( 3 ) , epoxiconazole ( 5 ) , penconazole ( 6 ) , triadimefon ( 9 ) were firstly eluted from both columns, the elution orders identified with an optical rotation detector didn't change with variety of column particles and mobile phases (acetronitrile/water and methanol/water). The plots of natural logarithms of the selectivity factors (ln α) for all fungicides except penconazole ( 6 ) versus the inverse of temperature (1/T) were linear in range of 5–40°C. The thermodynamic parameters (ΔH°, ΔS°, ΔΔH° and ΔΔS°) were calculated using Van't Hoff equations to understand the thermosynamic driving forces for enantioseparation. This work will be very helpful to obtain good enantiomeric separation and establish more efficient analytical method for triazole fungicides. Chirality, 2011. © 2011 Wiley‐Liss, Inc.     

Ly6C+Ly6G− Myeloid‐derived suppressor cells play a critical role in the resolution of acute inflammation and the subsequent tissue repair process after spinal cord injury

Acute inflammation is a prominent feature of central nervous system (CNS) insult and is detrimental to the CNS tissue. Although this reaction spontaneously diminishes within a short period of time, the mechanism underlying this inflammatory resolution remains largely unknown. In this study, we demonstrated that an initial infiltration of Ly6C⁺Ly6G^? immature monocyte fraction exhibited the same characteristics as myeloid‐derived suppressor cells (MDSCs), and played a critical role in the resolution of acute inflammation and in the subsequent tissue repair by using mice spinal cord injury (SCI) model. Complete depletion of Ly6C⁺Ly6G^? fraction prior to injury by anti‐Gr‐1 antibody (clone: RB6‐8C5) treatment significantly exacerbated tissue edema, vessel permeability, and hemorrhage, causing impaired neurological outcomes. Functional recovery was barely impaired when infiltration was allowed for the initial 24 h after injury, suggesting that MDSC infiltration at an early phase is critical to improve the neurological outcome. Moreover, intraspinal transplantation of ex vivo‐generated MDSCs at sites of SCI significantly reduced inflammation and promoted tissue regeneration, resulting in better functional recovery. Our findings reveal the crucial role of an Ly6C⁺Ly6G^? fraction as MDSCs in regulating inflammation and tissue repair after SCI, and also suggests an MDSC‐based strategy that can be applied to acute inflammatory diseases.     

Chromium ions inactivate electron transport and enhance superoxide generation in vivo in pea (Pisum sativum L. cv. Azad) root mitochondria

The effect in vivo of hexavalent chromium (Cr⁶⁺) on the respiratory electron transport activity and production of superoxide (O₂^–) radicals, was studied in submitochondrial particles (SMPs) prepared from mitochondria isolated from roots of 15‐day‐old pea (Pisum sativum L. cv. Azad) plants exposed to environmentally relevant (20 µm ) and acute (200 µm ) concentrations of chromium for 7 d. A concentration ‐dependent inactivation of electron transport activity from both NADH to O₂ (NADH oxidase) and succinate to O₂ (succinate oxidase) was observed. The electron transport activity was more sensitive to Cr⁶⁺ with NADH as the substrate than with succinate as the substrate. Although NADH dehydrogenase and succinate dehydrogenase were less affected, NADH: cytochrome c oxidoreductase and succinate: cytochrome c oxidoreductase activities were prominently affected by Cr⁶⁺. Cytochrome oxidase was the most susceptible complex of mitochondrial membranes to Cr⁶⁺, exhibiting maximal inactivation of activity both at 20 and 200 µm chromium concentrations. Cr⁶⁺ increased the generation of O₂^– radicals. This effect was more evident at 200 than at 20 µm . A significant increase in lipid peroxidation of mitochondrial membranes at 200 µm Cr⁶⁺ was the physiological impact of the metal‐induced enhanced generation of O₂^– radicals. An increase in superoxide dismutase (SOD) activity at 20 µm Cr⁶⁺ towards enhanced production of O₂^– radicals appeared to be a defence response in pea root mitochondria that, however, could not be sustained at 200 µm Cr⁶⁺. The results obtained concerning inactivation of mitochondrial electron transport and subsequent enhancement in the generation of O₂^– radicals suggest that root mitochondria are an important target of Cr⁶⁺‐induced oxidative stress in pea.     

HPLC purification and re‐evaluation of chemical identity of two circular bacteriocins,gassericin A and reutericin 6

Aim: The study aimed for the complete purification and recharacterization of the highly hydrophobic circular bacteriocins, gassericin A and reutericin 6. Methods and Results: Gassericin A and reutericin 6 were purified to homogeneity using previously described method and reverse‐phase HPLC with an octyl column and eluents of aqueous acetonitrile and 2‐propanol. Mass analysis, N‐terminal sequencing and bacteriocin assay of the HPLC‐purified bacteriocins showed the two bacteriocins had identical seamless circular structures with the same m/z value (5651) of [M + H]⁺ and both had the same specific activity. d/l‐ amino acid composition analysis using two distinct methods with the chiral fluorescent derivatization reagents (+)‐1‐(9‐fluorenyl)ethyl chloroformate and o‐phthalaldehyde/N‐acetyl‐l ‐cystein revealed neither gassericin A nor reutericin 6 contained d ‐alanine residues contrary to our previous results. Conclusion: Purified gassericin A and reutericin 6 are chemically identical circular molecules containing no d ‐alanine residues. Significance and Impact of the Study: The HPLC conditions developed in this study will facilitate advanced purification and correct characterization of other highly hydrophobic bacteriocins.