Application of Vitreoscilla Hemoglobin as a Green and Efficient Biocatalyst for the Synthesis of Benzoxazoles in Water.

We report an efficient and eco-friendly method for the Vitreoscilla hemoglobin (VHb)-catalyzed synthesis of benzoxazoles in water at room temperature. tert-Butyl hydroperoxide and 2,2,6,6-tetramethyl-1-piperidinyloxy were used as oxidant and radical scavenger, respectively. A total of 27 functionally diverse benzoxazoles were prepared in moderate to high yields (62 %-94 %) by the annulation reaction of phenols with amines in the presence of VHb in 1 h. Thus, this method is highly viable for practical applications. This work broadens the application of hemoglobin to organic synthesis.

Lipase-Catalyzed Phospha-Michael Addition Reactions under Mild Conditions.

Organophosphorus compounds are the core structure of many active natural products. The synthesis of these compounds is generally achieved by metal catalysis requiring specifically functionalized substrates or harsh conditions. Herein, we disclose the phospha-Michael addition reaction of biphenyphosphine oxide with various substituted ß-nitrostyrenes or benzylidene malononitriles. This biocatalytic strategy provides a direct route for the synthesis of C-P bonds with good functional group compatibility and simple and practical operation. Under the optimal conditions (styrene (0.5 mmol), biphenyphosphine oxide (0.5 mmol), Novozym 435 (300 U), and EtOH (1 mL)), lipase leads to the formation of organophosphorus compounds in yields up to 94% at room temperature. Furthermore, we confirm the role of the catalytic triad of lipase in this phospha-Michael addition reaction. This new biocatalytic system will have broad applications in organic synthesis.

Directed evolution of Escherichia coli surface-displayed Vitreoscilla hemoglobin as an artificial metalloenzyme for the synthesis of 5-imino-1,2,4-thiadiazoles.

Artificial metalloenzymes (ArMs) are constructed by anchoring organometallic catalysts to an evolvable protein scaffold. They present the advantages of both components and exhibit considerable potential for the in vivo catalysis of new-to-nature reactions. Herein, Escherichia coli surface-displayed Vitreoscilla hemoglobin (VHbSD-Co) that anchored the cobalt porphyrin cofactor instead of the original heme cofactor was used as an artificial thiourea oxidase (ATOase) to synthesize 5-imino-1,2,4-thiadiazoles. After two rounds of directed evolution using combinatorial active-site saturation test/iterative saturation mutagenesis (CAST/ISM) strategy, the evolved six-site mutation VHbSD-Co (6SM-VHbSD-Co) exhibited significant improvement in catalytic activity, with a broad substrate scope (31 examples) and high yields with whole cells. This study shows the potential of using VHb ArMs in new-to-nature reactions and demonstrates the applicability of E. coli surface-displayed methods to enhance catalytic properties through the substitution of porphyrin cofactors in hemoproteins in vivo.

Engineering of Dual-Function Vitreoscilla Hemoglobin: A One-Pot Strategy for the Synthesis of Unnatural α-Amino Acids.

Despite a well-developed and growing body of work on the directed evolution of hemoproteins, the potential of hemoproteins to catalyze non-natural reactions remains underexplored. This paper reports a new biocatalytic strategy for the one-pot synthesis of unnatural α-amino acids. Engineered variants of dual-function Vitreoscilla hemoglobin were found to efficiently catalyze N-H insertion and C-H sp3 alkylation, providing moderate to excellent yields (57%-95%) of unnatural α-amino acid derivatives and turnover numbers (1425-2375).

Over 20% of human transcripts might form sense-antisense pairs.

The major challenge to identifying natural sense- antisense (SA) transcripts from public databases is how to determine the correct orientation for an expressed sequence, especially an expressed sequence tag sequence. In this study, we established a set of very stringent criteria to identify the correct orientation of each human transcript. We used these orientation-reliable transcripts to create 26 741 transcription clusters in the human genome. Our analysis shows that 22% (5880) of the human transcription clusters form SA pairs, higher than any previous estimates. Our orientation-specific RT-PCR results along with the comparison of experimental data from previous studies confirm that our SA data set is reliable. This study not only demonstrates that our criteria for the prediction of SA transcripts are efficient, but also provides additional convincing data to support the view that antisense transcription is quite pervasive in the human genome. In-depth analyses show that SA transcripts have some significant differences compared with other types of transcripts, with regard to chromosomal distribution and Gene Ontology-annotated categories of physiological roles, functions and spatial localizations of gene products.

The role of segmental nodes in the pathological staging of non-small cell lung cancer.

BACKGROUND: Segmental nodes are not examined routinely in current clinical practice for lung cancer, the role of segmental nodes in pathological staging of non-small cell lung cancer after radical resection was investigated. METHODS: A total of 113 consecutive non-small cell lung cancer patients who underwent radical resection between June 2009 and December 2011 were retrospectively reviewed. All the operations were performed by the same group of surgeons. N2 nodes, hilar nodes, interlobar nodes and some lobar nodes were collected during surgery. The removed lung lobes were dissected routinely along lobar and segmental bronchi to collect lobar nodes and segmental nodes. The collected lymph nodes were separately labeled for histological examination. RESULTS: The detection rates of hilar nodes, interlobar nodes, lobar nodes and segmental nodes were 61.1%, 85.0%, 75.2% and 80.5%, respectively. The metastasis rates of hilar nodes, interlobar nodes, lobar nodes and segmental nodes were 5.3%, 10.5%, 16.8% and 14.2%, respectively. There were 68 cases of N0 disease, 16 cases of N1 disease and 29 cases of N2 disease. If an analysis of segmental lymph nodes had been omitted, six patients (37.5% of N1 disease) would have been down-staged to N0, and two cases of multiple-zone N1 disease would have been misdiagnosed as single-zone N1 disease, one patient would have been misdiagnosed as N2 disease with skip metastases. CONCLUSION: Segmental nodes play an important role in the accurate staging of non-small cell lung cancer, and routinely dissecting the segmental bronchi to collect the lymph nodes is feasible and may be necessary.

Large-scale protein annotation through gene ontology.

Recent progress in genomic sequencing, computational biology, and ontology development has presented an opportunity to investigate biological systems from a unique perspective, that is, examining genomes and transcriptomes through the multiple and hierarchical structure of Gene Ontology (GO). We report here our development of GO Engine, a computational platform for GO annotation, and analysis of the resultant GO annotations of human proteins. Protein annotation was centered on sequence homology with GO-annotated proteins and protein domain analysis. Text information analysis and a multiparameter cellular localization predictive tool were also used to increase the annotation accuracy, and to predict novel annotations. The majority of proteins corresponding to full-length mRNA in GenBank, and the majority of proteins in the NR database (nonredundant database of proteins) were annotated with one or more GO nodes in each of the three GO categories. The annotations of GenBank and SWISS-PROT proteins are available to the public at the GO Consortium web site.

Computational analysis of alternative splicing using EST tissue information.

Expressed sequence tags (ESTs) from normal and tumor tissues have been deposited in public databases. These ESTs and all mRNA sequences were aligned with the human genome sequence using LEADS, Compugen's alternative splicing modeling platform. We developed a novel computational approach to analyze tissue information of aligned ESTs in order to identify cancer-specific alternative splicing and gene segments highly expressed in particular cancers. Several genes, including one encoding a possible pre-mRNA splicing factor, displayed cancer-specific alternative splicing. In addition, multiple candidate gene segments highly expressed in colon cancers were identified.

Bridging expressed sequence alignments through targeted cDNA sequencing.

One of the major challenges in genome research is the identification of the complete set of genes in a genome. Alignments of expressed sequences (RNA and EST) with genomic sequences have been used to characterize genes. However, the number of alignments far exceeds the likely number of genes in a genome, suggesting that, for many genes, two or more alignments can be joined through overlapping sequences to yield accurate gene structures. High-throughput EST sequencing becomes less efficient in closing those alignment gaps due to its nonselective nature. We sought to bridge these alignments through a novel approach: targeted cDNA sequencing. Human expressed sequences from GenBank version 124 were aligned with the genomic sequence from NCBI build 24 using LEADS, Compugen's EST and RNA clustering and assembly software system. Nine hundred forty-eight pairs of alignments were selected based on EST clone information and/or their homology to the same known proteins. Reverse transcriptase PCR and sequencing yielded sequences for 363 of those pairs. These sequences helped characterize over 60 novel or otherwise incomplete genes in the recent UniGene build 153, which included over 1 million additional ESTs. These results indicate that this integrated and targeted strategy, combining computational prediction and experimental cDNA sequencing, can efficiently generate the overlapping sequences and enable the full characterization of genomes. Additional information about the contig pairs, the resultant overlapping sequences, tissue sources, and tissue profiles are available in a supplemental file.