Genome-wide identification of 5-methylcytosine sites in bacterial genomes by high-throughput sequencing of MspJI restriction fragments

Single-molecule Real-Time (SMRT) sequencing can easily identify sites of N6-methyladenine and N4-methylcytosine within DNA sequences, but similar identification of 5-methylcytosine sites is not as straightforward. In prokaryotic DNA, methylation typically occurs within specific sequence contexts, or motifs, that are a property of the methyltransferases that “write” these epigenetic marks. We present here a straightforward, cost-effective alternative to both SMRT and bisulfite sequencing for the determination of prokaryotic 5-methylcytosine methylation motifs. The method, called MFRE-Seq, relies on excision and isolation of fully methylated fragments of predictable size using MspJI-Family Restriction Enzymes (MFREs), which depend on the presence of 5-methylcytosine for cleavage. We demonstrate that MFRE-Seq is compatible with both Illumina and Ion Torrent sequencing platforms and requires only a digestion step and simple column purification of size-selected digest fragments prior to standard library preparation procedures. We applied MFRE-Seq to numerous bacterial and archaeal genomic DNA preparations and successfully confirmed known motifs and identified novel ones. This method should be a useful complement to existing methodologies for studying prokaryotic methylomes and characterizing the contributing methyltransferases.

Novel Intensified Alternatives for Purification of Levulinic Acid Recovered from Lignocellulosic Biomass

The development of a bio-based economy has its foundations in the development of efficient processes to optimize biomass potential. In this context there are a multitude of molecules that can be either synthetized or recovered from biomass, among those the so-called 12 building-blocks reported by the US Department of Energy. Even if their identification and importance is clearly defined, research efforts concerning the purification or separation of these platform molecules are limited. To fill this gap, different configurations for the purification of levulinic acid recovered from lignocellulosic biomass are examined and compared in this work. In particular, hybrid configurations obtained by the combination of liquid-liquid extraction and distillation have been considered. It was demonstrated how a deep understanding of the subspace including all extraction-assisted simple column distillation configurations represents a fundamental step in the synthesis of different process alternatives. From a separation efficiency and economic standpoint, the proposed intensified liquid-liquid thermally equivalent configuration (LL-TE) and liquid-liquid side stream column configuration (LL-SSC) are promising solutions. Nonetheless, their performances are deeply interrelated to the purity target defined by the designer.

Genome-Wide Identification of 5-Methylcytosine Sites in Bacterial Genomes By High-Throughput Sequencing of MspJI Restriction Fragments

ABSTRACTSingle-molecule Real-Time (SMRT) sequencing can easily identify sites of N6-methyladenine and N4-methylcytosine within DNA sequences, but similar identification of 5-methylcytosine sites is not as straightforward. In prokaryotic DNA, methylation typically occurs within specific sequence contexts, or motifs, that are a property of the methyltransferases that “write” these epigenetic marks. We present here a straightforward, cost-effective alternative to both SMRT and bisulfite sequencing for the determination of prokaryotic 5-methylcytosine methylation motifs. The method, called MFRE-Seq, relies on excision and isolation of fully methylated fragments of predictable size using MspJI-Family Restriction Enzymes (MFREs), which depend on the presence of 5-methylcytosine for cleavage. We demonstrate that MFRE-Seq is compatible with both Illumina and Ion Torrent sequencing platforms and requires only a digestion step and simple column purification of size-selected digest fragments prior to standard library preparation procedures. We applied MFRE-Seq to numerous bacterial and archaeal genomic DNA preparations and successfully confirmed known motifs and identified novel ones. This method should be a useful complement to existing methodologies for studying prokaryotic methylomes and characterizing the contributing methyltransferases.

Synthesis of Natural (−)-Antrocin and Its Enantiomer via Stereoselective Aldol Reaction

The total synthesis of (−)-antrocin and its enantiomer are presented. Antrocin (−)-1 is an important natural product which acts as an antiproliferative agent in a metastatic breast cancer cell line (IC50: 0.6 μM). The key features of this synthesis are: (a) selective anti-addition of trimethylsilyl cyanide (TMSCN) to α,β-unsaturated ketone; (b) resolution of (±)-7 using chiral auxiliary L-dimethyl tartrate through formation of cyclic ketal diastereomers followed by simple column chromatography separation and acid hydrolysis; (c) substrate-controlled stereoselective aldol condensation of (+)-12 with monomeric formaldehyde and pyridinium chlorochromate (PCC) oxidation for synthesis of essential lactone core in (−)-14; and (d) non-basic Lombardo olefination of the carbonyl at the final step to yield (−)-antrocin. In addition, (+)-9 cyclic ketal diastereomer was converted to (+)-antrocin with similar reaction sequences.

Utilization of Dairy Wastewater for Pollutants Removal and High Lipid Biomass Production by a Newly Isolated Microalgal Strains Chloromonas playfairii and Desmodesmus opoliensis

Two new unicellular microalgal species from milk processing unit situated at Anand, India were screened and tested for their growth rate and nutrient removal capability under dairy wastewater at outdoor conditions in a simple column aeration photobioreactor. Results of 18s rRNA sequence analysis indicated that these strains have a close relationship with Chloromonas playfairii and Desmodesmus opoliensis named as SBC 2 and SBC 3, respectively. Both the strains removed more than 92% of ammonical nitrogen and 99% of total phosphorus, whereas COD and BOD reductions were found to be more than 95%. Maximum biomass production was 1.7 and 1.2 g L-1 and lipid content was 15 and 12%, respectively from SBC 2 and SBC 3 after 15 d cultivation. This study demonstrates the potential of algae based biofuel production by coupling wastewater treatment with microalgae cultivation for high lipid containing biomass production using Chloromonas playfairii and Desmodesmus opoliensis.Int J Appl Sci Biotechnol, Vol 3(4): 699-707

A novel chemiluminescence sensor for determination of vanillin with magnetite–graphene oxide molecularly imprinted polymers

A simple column-packing method and a novel chemiluminescence sensor based on magnetite–graphene oxide molecularly imprinted polymers for vanillin determination.