Combining functional metagenomics and glycoanalytics to identify enzymes that facilitate structural characterization of sulfated N-glycans

Background Sulfate modification of N-glycans is important for several biological functions such as clearance of pituitary hormones or immunoregulation. Yet, the prevalence of this N-glycan modification and its functions remain largely unexplored. Characterization of N-glycans bearing sulfate modifications is hampered in part by a lack of enzymes that enable site-specific detection of N-glycan sulfation. In this study, we used functional metagenomic screening to identify enzymes that act upon sulfated N-acetylglucosamine (GlcNAc). Using multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (xCGE-LIF) -based glycoanalysis we proved their ability to act upon GlcNAc-6-SO4 on N-glycans. Results Our screen identified a sugar-specific sulfatase that specifically removes sulfate from GlcNAc-6-SO4 when it is in a terminal position on an N-glycan. Additionally, in the absence of calcium, this sulfatase binds to the sulfated glycan but does not remove the sulfate group, suggesting it could be used for selective isolation of sulfated N-glycans. Further, we describe isolation of a sulfate-dependent hexosaminidase that removes intact GlcNAc-6-SO4 (but not asulfated GlcNAc) from a terminal position on N-glycans. Finally, the use of these enzymes to detect the presence of sulfated N-glycans by xCGE-LIF is demonstrated. Conclusion The present study demonstrates the feasibility of using functional metagenomic screening combined with glycoanalytics to discover enzymes that act upon chemical modifications of glycans. The discovered enzymes represent new specificities that can help resolve the presence of GlcNAc-6-SO4 in N-glycan structural analyses.

Study of Aggregation Behavior of Predesigned Azobenzene-Cholesteryl Derivatives in Deep Eutectic Solvents

Structurally isomeric cholesteryl-appended azobenzene derivatives (azo-1 to azo-5) with various substituents, such as H/unsubstituted, ether, ester, and nitro at the terminal position of azobenzene units were designed, and synthesized. The gelation ability and aggregation behavior of the above synthesized azobenzene-cholesteryl derivatives in deep eutectic solvents (DES) such as Zinc Chloride: Ethylene Glycol (Zn:EG), Choline Chloride: EG (Ch:EG), Choline Chloride: Urea (Ch: Urea), and Choline Chloride: Glycine (Ch: Gly) were studied. The results revealed that all the azo derivatives formed semi-transparent and strong/hard eutectic gels in at least one DES except azo-4 which formed gel in two DES. The morphological analyses by scanning electron microscopy (SEM) exhibited entangled dense fibrous, flowers, and sheet-like textures, depending on the nature of DES as well as azo derivatives. Like all azobenzene-based organo-gelators, UV-triggered gel-to-sol transition was expected for these eutectic gels. However, these eutectic gels did not undergo the gel-to-sol transition under UV irradiation. This could be due to the hardness of the gel, which arrests the structural transformation from trans-to-cis during photolysis. It was further confirmed by absorption profiles of before and after irradiation of eutectic gels. Regarding application, an attempt has been made to use eutectic gels as a template for the synthesis of nanomaterials and the results revealed that the azo-4 gel can be used to prepare aggregated highly dense nanorods of copper chloride.

The Sprague-Grundy Functions of Saturations of Misère Nim

We consider misère Nim as a normal-play game obtained from Nim by removing the terminal position. While explicit formulas are known for the Sprague-Grundy functions of Nim and Welter's game, no explicit formula is known for that of misère Nim. All three of these games can be considered as position restrictions of Nim. What are the differences between them? We point out that Nim and Welter's game are saturated, but misère Nim is not. Moreover, we present explicit formulas for the Sprague-Grundy functions of saturations of misère Nim, which are obtained from misère Nim by adjoining some moves.

Existence results for a coupled system of Caputo type fractional integro-differential equations with multi-point and sub-strip boundary conditions

This paper is concerned with the existence and uniqueness of solutions for a coupled system of Liouville–Caputo type fractional integro-differential equations with multi-point and sub-strip boundary conditions. The fractional integro-differential equations involve Caputo derivative operators of different orders and finitely many Riemann–Liouville fractional integral and non-integral type nonlinearities. The boundary conditions at the terminal position $t=1$ t = 1 involve sub-strips and multi-point contributions. The Banach fixed point theorem and the Leray–Schauder alternative are used to establish our results. The obtained results are illustrated with the aid of examples.

Click Beetle Mitogenomics with the Definition of a New Subfamily Hapatesinae from Australasia (Coleoptera: Elateridae)

Elateridae is a taxon with very unstable classification and a number of conflicting phylogenetic hypotheses have been based on morphology and molecular data. We assembled eight complete mitogenomes for seven elaterid subfamilies and merged these taxa with an additional 22 elaterids and an outgroup. The structure of the newly produced mitogenomes showed a very similar arrangement with regard to all earlier published mitogenomes for the Elateridae. The maximum likelihood and Bayesian analyses indicated that Hapatesus Candèze, 1863, is a sister of Parablacinae and Pityobiinae. Therefore, Hapatesinae, a new subfamily, is proposed for the Australian genera Hapatesus (21 spp.) and Toorongus Neboiss, 1957 (4 spp.). Parablacinae, Pityobiinae, and Hapatesinae have a putative Gondwanan origin as the constituent genera are known from the Australian region (9 genera) and Neotropical region (Tibionema Solier, 1851), and only Pityobius LeConte, 1853, occurs in the Nearctic region. Another putative Gondwanan lineage, the Afrotropical Morostomatinae, forms either a serial paraphylum with the clade of Parablacinae, Pityobiinae, and Hapatesinae or is rooted in a more terminal position, but always as an independent lineage. An Eudicronychinae lineage was either recovered as a sister to Melanotini or as a deep split inside Elaterinae and we herein transfer the group to Elaterinae as Eudicronychini, a new status. The mitochondrial genomes provide a sufficient signal for the placement of most lineages, but the deep bipartitions need to be compared with phylogenomic analyses.

Tuning G-Quadruplex Nanostructures with Lipids. Towards Designing Hybrid Scaffolds for Oligonucleotide Delivery

Two G-quadruplex forming oligonucleotides [d(TG4T)4 and d(TG6T)4] were selected as two tetramolecular quadruplex nanostructures because of their demonstrated ability to be modified with hydrophobic molecules. This allowed us to synthesize two series of G-quadruplex conjugates that differed in the number of G-tetrads, as well as in the terminal position of the lipid modification. Both solution and solid-phase syntheses were carried out to yield the corresponding lipid oligonucleotide conjugates modified at their 3′- and 5′-termini, respectively. Biophysical studies confirmed that the presence of saturated alkyl chains with different lengths did not affect the G-quadruplex integrity, but increased the stability. Next, the G-quadruplex domain was added to an 18-mer antisense oligonucleotide. Gene silencing studies confirmed the ability of such G-rich oligonucleotides to facilitate the inhibition of target Renilla luciferase without showing signs of toxicity in tumor cell lines.

Reconstructed evolutionary history of the yeast septins Cdc11 and Shs1

Septins are GTP-binding proteins conserved across metazoans. They can polymerize into extended filaments and, hence, are considered a component of the cytoskeleton. The number of individual septins varies across the tree of life—yeast (Saccharomyces cerevisiae) has seven distinct subunits, a nematode (Caenorhabditis elegans) has two, and humans have 13. However, the overall geometric unit (an apolar hetero-octameric protomer and filaments assembled there from) has been conserved. To understand septin evolutionary variation, we focused on a related pair of yeast subunits (Cdc11 and Shs1) that appear to have arisen from gene duplication within the fungal clade. Either Cdc11 or Shs1 occupies the terminal position within a hetero-octamer, yet Cdc11 is essential for septin function and cell viability, whereas Shs1 is not. To discern the molecular basis of this divergence, we utilized ancestral gene reconstruction to predict, synthesize, and experimentally examine the most recent common ancestor (“Anc.11-S”) of Cdc11 and Shs1. Anc.11-S was able to occupy the terminal position within an octamer, just like the modern subunits. Although Anc.11-S supplied many of the known functions of Cdc11, it was unable to replace the distinct function(s) of Shs1. To further evaluate the history of Shs1, additional intermediates along a proposed trajectory from Anc.11-S to yeast Shs1 were generated and tested. We demonstrate that multiple events contributed to the current properties of Shs1: (1) loss of Shs1–Shs1 self-association early after duplication, (2) co-evolution of heterotypic Cdc11–Shs1 interaction between neighboring hetero-octamers, and (3) eventual repurposing and acquisition of novel function(s) for its C-terminal extension domain. Thus, a pair of duplicated proteins, despite constraints imposed by assembly into a highly conserved multi-subunit structure, could evolve new functionality via a complex evolutionary pathway.

Evolutionary dynamics of rDNA clusters on chromosomes of buthid scorpions (Chelicerata: Arachnida)

We examined the distribution of genes for major ribosomal RNAs (rDNA) on holokinetic chromosomes of 74 species belonging to 19 genera of scorpions from the family Buthidae using fluorescence in situ hybridization (FISH). Our analysis revealed differences between the two main evolutionary lineages within the family. The genera belonging to the ‘Buthus group’, with a proposed Laurasian origin, possess one pair of rDNA mainly in an interstitial position, with the only exceptions being the terminal location found in some Hottentotta and Buthacus species, possibly as a result of chromosome fissions. All the remaining buthid ‘groups’ possess rDNA found strictly in a terminal position. However, the number of signals may increase from an ancestral state of one pair of rDNA loci to up to seven signals in Reddyanus ceylonensis Kovařík et al., 2016. Despite the differences in evolutionary dynamics of the rDNA clusters between the ‘Buthus group’ and other lineages investigated, we found a high incidence of reciprocal translocations and presence of multivalent associations during meiosis in the majority of the genera studied. These phenomena seem to be typical for the whole family Buthidae.