An SRLLR motif downstream of the scissile bond enhances enterokinase cleavage efficiency

In a previous paper, we reported more efficient enterokinase cleavage at a C-terminal non-target LKGDR(201) site compared with an internally sited canonical recognition site, DDDDK(156). When this non-target site was placed internally to replace DDDDK(156) between the thioredoxin moiety and mouse NT-proCNP(1-50), this site was poorly processed leading us to conclude that efficient processing at LKGDR(201) in the first instance was due to its accessibility at the C-terminus of the fusion protein. Subsequently, we reasoned that treatment of thioredoxin-fused NT-proCNP(1-81) would allow us to retrieve full-length NT-proCNP(1-81) without undue processing at the LKGDR(201) site since this non-target site would now be located internally about 36 residues away from the C-terminus and hence not be hydrolyzed efficiently. Surprisingly, ESI-MS data showed that the LKGDR site in thioredoxin-fused human NT-proCNP(1-81) was still very efficiently cleaved and revealed a new but slow hydrolysis site with the sequence RVDTK/SRAAW to yield a peptide consistent with NT-proCNP(58-81). The evidence obtained from these experiments led us to postulate that efficient cleavage at the non-target LKGDR(201) site was not merely influenced by steric constraints but also by the sequence context downstream of the scissile bond. Hence, we constructed variants of thioredoxin-mouse NT-proCNP(1-50) where SRLLR residues (i.e. those immediately downstream from the LKGDR(201) site in NT-proCNP(1-50)) were systematically added one at a time downstream of the internal DDDDK(156) site. To evaluate the relative effects of site accessibility and downstream sequence context on the efficiency of enterokinase cleavage, we have also replaced the native LKGDR(201) sequence with DDDDK(201). Our results showed that incremental addition of SRLLR residues led to a steady increase in the rate of hydrolysis at DDDDK(156). Further variants comprising DDDDK(156)SS, DDDDK(156)SD and DDDDK(156)RR showed that the minimal critical determinants for enhanced enterokinase cleavage are serine in the P1' position followed by a serine or a basic residue, lysine or arginine, in the P2' position. Our data provided conclusive evidence that the influence of downstream sequences on recombinant light chain enterokinase activity was greater than accessibility of the target site at the terminus region of the protein. We further showed that the catalytic efficiency of the native holoenzyme was influenced primarily by residues on the N-terminal side of the scissile bond while being neutral to residues on the C-terminal side. Finally, we found that cleavage of all nine fusion proteins reflects accurate hydrolysis at the DDDDK(156) and DDDDK(201) sites when recombinant light chain enterokinase was used while non-specific processing at secondary sites were observed when these fusion proteins were treated with the native holoenzyme.     

Epidemiological and Virological Characteristics of Influenza Viruses Circulating in Cambodia from 2009 to 2011

Background

The Cambodian National Influenza Center (NIC) monitored and characterized circulating influenza strains from 2009 to 2011.

Methodology/Principal Findings

Sentinel and study sites collected nasopharyngeal specimens for diagnostic detection, virus isolation, antigenic characterization, sequencing and antiviral susceptibility analysis from patients who fulfilled case definitions for influenza-like illness, acute lower respiratory infections and event-based surveillance. Each year in Cambodia, influenza viruses were detected mainly from June to November, during the rainy season. Antigenic analysis show that A/H1N1pdm09 isolates belonged to the A/California/7/2009-like group. Circulating A/H3N2 strains were A/Brisbane/10/2007-like in 2009 before drifting to A/Perth/16/2009-like in 2010 and 2011. The Cambodian influenza B isolates from 2009 to 2011 all belonged to the B/Victoria lineage represented by the vaccine strains B/Brisbane/60/2008 and B/Malaysia/2506/2004. Sequences of the M2 gene obtained from representative 2009–2011 A/H3N2 and A/H1N1pdm09 strains all contained the S31N mutation associated with adamantanes resistance except for one A/H1N1pdm09 strain isolated in 2011 that lacked this mutation. No reduction in the susceptibility to neuraminidase inhibitors was observed among the influenza viruses circulating from 2009 to 2011. Phylogenetic analysis revealed that A/H3N2 strains clustered each year to a distinct group while most A/H1N1pdm09 isolates belonged to the S203T clade.

Conclusions/Significance

In Cambodia, from 2009 to 2011, influenza activity occurred throughout the year with peak seasonality during the rainy season from June to November. Seasonal influenza epidemics were due to multiple genetically distinct viruses, even though all of the isolates were antigenically similar to the reference vaccine strains. The drug susceptibility profile of Cambodian influenza strains revealed that neuraminidase inhibitors would be the drug of choice for influenza treatment and chemoprophylaxis in Cambodia, as adamantanes are no longer expected to be effective.     

Rational Engineering and Characterization of an mAb that Neutralizes Zika Virus by Targeting a Mutationally Constrained Quaternary Epitope

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The role of sequence context,nucleotide pool balance and stress in 2′-deoxynucleotide misincorporation in viral,bacterial and mammalian RNA

The misincorporation of 2′-deoxyribonucleotides (dNs) into RNA has important implications for the function of non-coding RNAs, the translational fidelity of coding RNAs and the mutagenic evolution of viral RNA genomes. However, quantitative appreciation for the degree to which dN misincorporation occurs is limited by the lack of analytical tools. Here, we report a method to hydrolyze RNA to release 2′-deoxyribonucleotide-ribonucleotide pairs (dNrN) that are then quantified by chromatography-coupled mass spectrometry (LC-MS). Using this platform, we found misincorporated dNs occurring at 1 per 10³ to 10⁵ ribonucleotide (nt) in mRNA, rRNAs and tRNA in human cells, Escherichia coli, Saccharomyces cerevisiae and, most abundantly, in the RNA genome of dengue virus. The frequency of dNs varied widely among organisms and sequence contexts, and partly reflected the in vitro discrimination efficiencies of different RNA polymerases against 2′-deoxyribonucleoside 5′-triphosphates (dNTPs). Further, we demonstrate a strong link between dN frequencies in RNA and the balance of dNTPs and ribonucleoside 5′-triphosphates (rNTPs) in the cellular pool, with significant stress-induced variation of dN incorporation. Potential implications of dNs in RNA are discussed, including the possibilities of dN incorporation in RNA as a contributing factor in viral evolution and human disease, and as a host immune defense mechanism against viral infections.     

Identification of Molecular Markers Associated with Alteration of Receptor-Binding Specificity in a Novel Genotype of Highly Pathogenic Avian Influenza A(H5N1) Viruses Detected in Cambodia in 2013

Human infections with influenza A(H5N1) virus in Cambodia increased sharply during 2013. Molecular characterization of viruses detected in clinical specimens from human cases revealed the presence of mutations associated with the alteration of receptor-binding specificity (K189R, Q222L) and respiratory droplet transmission in ferrets (N220K with Q222L). Discovery of quasispecies at position 222 (Q/L), in addition to the absence of the mutations in poultry/environmental samples, suggested that the mutations occurred during human infection and did not transmit further.     

Influenza Vaccination Guidelines and Vaccine Sales in Southeast Asia: 2008–2011

Background

Southeast Asia is a region with great potential for the emergence of a pandemic influenza virus. Global efforts to improve influenza surveillance in this region have documented the burden and seasonality of influenza viruses and have informed influenza prevention strategies, but little information exists about influenza vaccination guidelines and vaccine sales.

Methods

To ascertain the existence of influenza vaccine guidelines and define the scope of vaccine sales, we sent a standard three-page questionnaire to the ten member nations of the Association of Southeast Asian Nations. We also surveyed three multinational manufacturers who supply influenza vaccines in the region.

Results

Vaccine sales in the private sector were <1000 per 100,000 population in the 10 countries. Five countries reported purchasing vaccine for use in the public sector. In 2011, Thailand had the highest combined reported rate of vaccine sales (10,333 per 100,000). In the 10 countries combined, the rate of private sector sales during 2010–2011 (after the A(H1N1)2009pdm pandemic) exceeded 2008 pre-pandemic levels. Five countries (Indonesia, Malaysia, Singapore, Thailand and Vietnam) had guidelines for influenza vaccination but only two were consistent with global guidelines. Four recommended vaccination for health care workers, four for elderly persons, three for young children, three for persons with underlying disease, and two for pregnant women.

Conclusions

The rate of vaccine sales in Southeast Asia remains low, but there was a positive impact in sales after the A(H1N1)2009pdm pandemic. Low adherence to global vaccine guidelines suggests that more work is needed in the policy arena.     

Signal Processing for Metagenomics: Extracting Information from the Soup

Traditionally, studies in microbial genomics have focused on single-genomes from cultured species, thereby limiting their focus to the small percentage of species that can be cultured outside their natural environment. Fortunately, recent advances in high-throughput sequencing and computational analyses have ushered in the new field of metagenomics, which aims to decode the genomes of microbes from natural communities without the need for cultivation. Although metagenomic studies have shed a great deal of insight into bacterial diversity and coding capacity, several computational challenges remain due to the massive size and complexity of metagenomic sequence data. Current tools and techniques are reviewed in this paper which address challenges in 1) genomic fragment annotation, 2) phylogenetic reconstruction, 3) functional classification of samples, and 4) interpreting complementary metaproteomics and metametabolomics data. Also surveyed are important applications of metagenomic studies, including microbial forensics and the roles of microbial communities in shaping human health and soil ecology.     

Combining gene prediction methods to improve metagenomic gene annotation

Background  

Traditional gene annotation methods rely on characteristics that may not be available in short reads generated from next generation technology, resulting in suboptimal performance for metagenomic (environmental) samples. Therefore, in recent years, new programs have been developed that optimize performance on short reads. In this work, we benchmark three metagenomic gene prediction programs and combine their predictions to improve metagenomic read gene annotation.