Binpairs: Utilization of Illumina Paired-End Information for Improving Efficiency of Taxonomic Binning of Metagenomic Sequences

Motivation

Paired-end sequencing protocols, offered by next generation sequencing (NGS) platforms like Illumia, generate a pair of reads for every DNA fragment in a sample. Although this protocol has been utilized for several metagenomics studies, most taxonomic binning approaches classify each of the reads (forming a pair), independently. The present work explores some simple but effective strategies of utilizing pairing-information of Illumina short reads for improving the accuracy of taxonomic binning of metagenomic datasets. The strategies proposed can be used in conjunction with all genres of existing binning methods.

Results

Validation results suggest that employment of these “Binpairs” strategies can provide significant improvements in the binning outcome. The quality of the taxonomic assignments thus obtained are often comparable to those that can only be achieved with relatively longer reads obtained using other NGS platforms (such as Roche).

Availability

An implementation of the proposed strategies of utilizing pairing information is freely available for academic users at https://metagenomics.atc.tcs.com/binning/binpairs.     

Specificity of Pyrrolysyl-tRNA Synthetase for Pyrrolysine and Pyrrolysine Analogs

Pyrrolysine, the 22nd amino acid, is encoded by amber (TAG = UAG) codons in certain methanogenic archaea and bacteria. PylS, the pyrrolysyl-tRNA synthetase, ligates pyrrolysine to tRNA^Pyl for amber decoding as pyrrolysine. PylS and tRNA^Pyl have potential utility in making tailored recombinant proteins. Here, we probed interactions necessary for recognition of substrates by archaeal PylS via synthesis of close pyrrolysine analogs and testing their reactivity in amino acid activation assays. Replacement of the methylpyrroline ring of pyrrolysine with cyclopentane indicated that solely hydrophobic interactions with the ring-binding pocket of PylS are sufficient for substrate recognition. However, a 100-fold increase in the specificity constant of PylS was observed with an analog, 2-amino-6-((R)-tetrahydrofuran-2-carboxamido)hexanoic acid (2Thf-lys), in which tetrahydrofuran replaced the pyrrolysine methylpyrroline ring. Other analogs in which the electronegative atom was moved to different positions suggested PylS preference for a hydrogen-bond-accepting group at the imine nitrogen position in pyrrolysine. 2Thf-lys was a preferred substrate over a commonly employed pyrrolysine analog, but the specificity constant for 2Thf-lys was 10-fold lower than for pyrrolysine itself, largely due to the change in K_m. The in vivo activity of the analogs in supporting UAG suppression in Escherichia coli bearing genes for PylS and tRNA^Pyl was similar to in vitro results, with l-pyrrolysine and 2Thf-lys supporting the highest amounts of UAG translation. Increasing concentrations of either PylS substrate resulted in a linear increase in UAG suppression, providing a facile method to assay bioactive pyrrolysine analogs. These results illustrate the relative importance of the H-bonding and hydrophobic interactions in the recognition of the methylpyrroline ring of pyrrolysine and provide a promising new series of easily synthesized pyrrolysine analogs that can serve as scaffolds for the introduction of novel functional groups into recombinant proteins.     

Botryococcus braunii: A Renewable Source of Hydrocarbons and Other Chemicals

ABSTRACT:?

Botryococcus braunii, a green colonial microalga, is an unusually rich renewable source of hydrocarbons and other chemicals. Hydrocarbons can constitute up to 75% of the dry mass of B. braunii. This review details the various facets of biotechnology of B. braunii, including its microbiology and physiology; production of hydrocarbons and other compounds by the alga; methods of culture; downstream recovery and processing of algal hydrocarbons; and cloning of the algal genes into other microorganisms. B. braunii converts simple inorganic compounds and sunlight to potential hydrocarbon fuels and feedstocks for the chemical industry. Microorganisms such as B. braunii can, in the long run, reduce our dependence on fossil fuels and because of this B. braunii continues to attract much attention.     

Nucleic acids binding strategies of small molecules: Lessons from alkaloids

Background

Nucleic acids are now important targets for therapeutic intervention. Alkaloids are an important class of molecules that have myriad therapeutic utility. Isoquinoline and benzophenanthridine alkaloids exhibit multiple pharmacological activities which are often related to their strong nucleic acid binding abilities. Therefore, a review of their interaction aspects with varying nucleic acid structures is essential for rational design and development as therapeutic agents.

Mangrove associates versus true mangroves: a comparative analysis of leaf litter decomposition in Sundarban

Mangrove species are broadly classified as ‘true mangroves’ and ‘mangrove associates’. We hypothesized that the leaf litter decomposition rates of true mangroves differ significantly from the mangrove associates under the same ecological and bio-climatic conditions. In order to test this hypothesis, the leaf litter decay rates of 24 true mangrove species and 10 mangrove associates along with the concomitant carbon and nitrogen dynamics of the litters were studied in the tropical mangrove forest of Sundarban by means of litter bags. The decomposition was monitored for six consecutive weeks in the pre-monsoon, monsoon and post-monsoon season. All the species in general went through a rapid decay phase in the first 2 weeks, however, the rate substantially decreased in the following 4 weeks. Most of the species studied had significant seasonal variability (p < 0.05) in the decay rate. Species-specific decay was highest throughout the monsoon and least during the post-monsoon season. The mean dry weight composition (i.e. percentage of dry weight of the leaf litters remaining at the end of weekly intervals) of the true mangroves was 10–12 % higher than the mangrove associates throughout the sampling period. The mean decay constants (K in week^?1) of the true mangroves were 0.15 ± 0.05, 0.20 ± 0.06 and 0.16 ± 0.05 in the pre-monsoon, monsoon and post-monsoon season respectively. The mangrove associates had significantly higher decay constants in the respective seasons that followed the order 0.23 ± 0.09, 0.25 ± 0.06 and 0.24 ± 0.09. As a consequence, the computed mean half-life period of the true mangrove litters (32 ± 11 days) was much higher than the mangrove associates (23 ± 11 days). This showed that collectively the leaf litters of mangrove associates degraded at a much faster rate than the true mangroves throughout the annual cycle and thus our hypothesis was justified.     

Membrane Vesicles of Group B Streptococcus Disrupt Feto-Maternal Barrier Leading to Preterm Birth

Infection of the genitourinary tract with Group B Streptococcus (GBS), an opportunistic gram positive pathogen, is associated with premature rupture of amniotic membrane and preterm birth. In this work, we demonstrate that GBS produces membrane vesicles (MVs) in a serotype independent manner. These MVs are loaded with virulence factors including extracellular matrix degrading proteases and pore forming toxins. Mice chorio-decidual membranes challenged with MVs ex vivo resulted in extensive collagen degradation leading to loss of stiffness and mechanical weakening. MVs when instilled vaginally are capable of anterograde transport in mouse reproductive tract. Intra-amniotic injections of GBS MVs in mice led to upregulation of pro-inflammatory cytokines and inflammation mimicking features of chorio-amnionitis; it also led to apoptosis in the chorio-decidual tissue. Instillation of MVs in the amniotic sac also resulted in intrauterine fetal death and preterm delivery. Our findings suggest that GBS MVs can independently orchestrate events at the feto-maternal interface causing chorio-amnionitis and membrane damage leading to preterm birth or fetal death.     

Revision of Gangesia (Cestoda: Proteocephalidea) in the Indomalayan Region: Morphology,Molecules and Surface Ultrastructure

Tapeworms of Gangesia Woodland, 1924 (Cestoda: Proteocephalidea) parasitic in freshwater fishes in the Indomalayan Region were critically reviewed. Evaluation of type specimens and newly collected materials from Bangladesh, Cambodia and India, as well as critical examination of extensive literature have shown that only the following four species, instead of 48 nominal species of Gangesia and Silurotaenia Nybelin, 1942 reported from this region (36 new synonymies proposed), are valid: Gangesia bengalensis (Southwell, 1913), type-species of the genus and most common parasite of Wallago attu (Siluridae), G. macrones Woodland, 1924 typical of Sperata seenghala (Bagridae), both species characterized by the possession of two circles of hooks on the rostellum-like organ and several rows of hooklets on the anterior margins of suckers; G. agraensis Verma, 1928 from W. attu (typical host), which has the scolex with only one circle of hooks and 1–3 incomplete rows of tiny hooklets on the suckers; and G. vachai (Gupta and Parmar, 1988) n. comb. from several catfishes, which possesses 4–6 circles of hooks and 5–11 rows of hooklets on the anterior half of suckers. Scolex morphology, including surface ultrastructure (microtriches), of all but one species (G. vachai) is described for the first time using scanning electron microscopy. A phylogenetic analysis based on the partial sequences encoding the large nuclear ribosomal subunit RNA gene has shown that three Indomalayan species, namely G. bengalensis, G. macrones and G. vachai, form a monophyletic group within Gangesia, whereas G. agraensis tends to form a clade with the Palaearctic species of the genus. A table with differential characters of all species from the Indomalayan Region is also provided together with a key to identification of genera of the subfamily Gangesiinae. The present study demonstrates that species of Silurotaenia do not occur in the Indomalayan region.     

Biophysical Characterization of Essential Phosphorylation at the Flexible C-Terminal Region of C-Raf with 14-3-3ζ Protein

Phosphorylation at the C-terminal flexible region of the C-Raf protein plays an important role in regulating its biological activity. Auto-phosphorylation at serine 621 (S621) in this region maintains C-Raf stability and activity. This phosphorylation mediates the interaction between C-Raf and scaffold protein 14-3-3ζ to activate the downstream MEK kinase pathway. In this study, we have defined the interaction of C-terminal peptide sequence of C-Raf with 14-3-3ζ protein and determined the possible structural adaptation of this region. Biophysical elucidation of the interaction was carried out using phosphopeptide (residue number 615–630) in the presence of 14-3-3ζ protein. Using isothermal titration calorimetry (ITC), a high binding affinity with micro-molar range was found to exist between the peptide and 14-3-3ζ protein, whereas the non-phosphorylated peptide did not show any appreciable binding affinity. Further interaction details were investigated using several biophysical techniques such as circular dichroism (CD), fluorescence, and nuclear magnetic resonance (NMR) spectroscopy, in addition to molecular modeling. This study provides the molecular basis for C-Raf C-terminal-derived phosphopeptide interaction with 14-3-3ζ protein as well as structural insights responsible for phosphorylated S621-mediated 14-3-3ζ binding at an atomic resolution.